Methods of generating nanoarrays and microarrays

ABSTRACT

The methods described herein provide a means of producing an array of spatially separated proteins. The method relies on covalently attaching each protein of the plurality of proteins to a structured nucleic acid particle (SNAP), and attaching the SNAPs to a solid support.

CROSS-REFERENCE

This application is a continuation of application Ser. No. 17/062,405,filed Oct. 2, 2020, which is a continuation of International ApplicationNo. PCT/US2019/025909, filed Apr. 4, 2019, which claims the benefit ofU.S. Provisional Application No. 62/652,849, filed Apr. 4, 2018, each ofwhich is incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 7, 2021, isnamed 51612-706_302_SL.txt and is 1,461 bytes in size.

BACKGROUND OF THE INVENTION

Microarrays and nanoarrays may be used for assessing biological andchemical entities. It may be beneficial to tailor the design ofnanoarrays and microarray to particular assessments.

SUMMARY OF THE INVENTION

The present disclosure provides methods and systems for separatingbiological and chemical entities.

An aspect of the invention provides a composition comprising astructured nucleic acid particle (SNAP) covalently attached to aprotein. In some cases, said SNAP is attached to a solid support. Insome cases, said SNAP is covalently attached to a solid support. In somecases, said SNAP is non-covalently attached to a solid support.

An aspect of the invention provides a composition comprising a SNAPcovalently attached to a biomolecule. In some cases, said SNAP isattached to a solid support. In some cases, said SNAP is covalentlyattached to a solid support. In some cases, said SNAP is non-covalentlyattached to a solid support.

An aspect of the invention provides a composition comprising a solidsupport attached to a plurality of structured nucleic acid particles(SNAPs), wherein each of said plurality of SNAPs is attached to abiomolecule. In some cases, the plurality of SNAPs are arranged in anarray.

An aspect of the invention provides a method of attaching a singleprotein to an attachment site on a solid support, wherein the attachmentsite is larger than the protein; the method comprising covalentlyattaching the protein to a SNAP; wherein the diameter of said SNAP is atleast as large as the diameter of the attachment site; and attaching theSNAP to the attachment site. In some cases, each of said plurality ofproteins is attached to an attachment site on said solid support; suchthat each attachment site is attached to a single protein.

An aspect of the invention provides a biomolecule array comprising aplurality of biomolecules attached to a solid support; wherein eachbiomolecule of the plurality of biomolecules is covalently attached to alinker and the linker is attached to the solid support; and wherein eachlinker is attached to only one biomolecule of the plurality ofbiomolecules and each linker has a diameter of at least 50 nm.

An aspect of the invention provides a method of producing an array ofspatially separated proteins from a plurality of proteins, the methodcomprising covalently attaching each protein of the plurality ofproteins to an end of a nucleic acid molecule comprising a SNAP, andattaching the SNAPs to a solid support, thereby producing an array ofspatially separated proteins.

An aspect of the invention provides a composition comprising a protein,a SNAP and a solid support; wherein the protein is covalently bound tothe SNAP, and wherein the protein does not contact the solid support.

An aspect of the invention provides a method of producing an array ofspatially separated biological or chemical entities, the methodcomprising: obtaining a solid support with attachment sites, obtaining asample comprising a plurality of biological or chemical entities,obtaining seeds, each with a functional group, covalently attaching eachbiological or chemical entity of the plurality of biological or chemicalentities to a single seed via the functional group, growing eachattached seed to a SNAP of desired size, attaching the SNAPs to theattachment sites of the array, thereby producing an array of spatiallyseparated biological or chemical entities.

In some cases, a solid support is a glass, silica, plastic, silicon,gold, metal, chromium, titanium, titanium oxide, tin, or tin oxidesupport. In some cases, a solid support is optically opaque. In somecases, the solid support is optically clear. In some cases, said solidsupport is modified to have a positive charge. In some cases, said solidsupport is modified to have a negative charge. In some cases, said solidsupport is modified to have functional groups which may bind the SNAPs.In some cases, said solid support comprises attachment sites which aremodified to be different to surrounding surfaces. In some cases, saidsolid support comprises an array of attachment sites. In some cases,each attachment site is at least 70 nm from each other attachment site.In some cases, each attachment site is at least 25 nm from each otherattachment site. In some cases, the distance between the edges of anytwo attachment sites is greater than the radius of the SNAP used. Insome cases, the distance between the edges of any two attachment sitesis greater than the diameter of the SNAP used.

In some cases, the molecules are proteins. In some cases, the seeds areoligonucleotides.

In some cases, the oligonucleotides are modified on the 3′ end with afunctional group. In some cases, the oligonucleotides are modified onthe 5′ end with a functional group. In some cases, the functional groupis selected from the group consisting of amines, thiols, carboxylicacids, triple bonds, double bonds, epoxides, alkynes, alkenes,cycloalkynes, azides, cyclo-octynes, cycloalkynes, norbornenes,tetrazines, cyclloctanes, epoxides, and hydroxyls. In some cases, theoligonucleotides are modified to comprise a photocleaveable bond. Insome cases, the SNAPs are formed by rolling circle amplification. Insome cases, the SNAPs are dendrimers. In some cases, the dendrimers arepositively charged and the attachment sites on the array are negativelycharged. In some cases, the dendrimers are negatively charged and theattachment sites on the array are positively charged. In some cases, theSNAPs have a diameter of approximately 100 nm. In some cases, the SNAPshave a diameter of approximately 300 nm. In some cases the SNAPs have adiameter of between about 10 nm and 500 m. In some cases, the SNAPs havea diameter of between about 10 nm and 50 m. In some cases, the SNAPshave a diameter of between about 10 nm and 5 m. In some cases, the SNAPshave a diameter of between about 100 nm and 500 nm. In some cases, SNAPsadhere to the solid support through an electrostatic interaction.

An aspect of the invention provides a method of achieving spatialseparation of molecules, the method comprising: obtaining a plurality ofmolecules, obtaining seeds, each with a functional group, covalentlyattaching each of the plurality of molecules to a single seed via thefunctional group, growing each attached seed to a SNAP of desired size,attaching the SNAPs to a solid support, thereby achieving spatialseparation of single molecules.

An aspect of the invention provides an array of single molecules, eachsingle molecule being attached to a SNAP of desired size, the SNAP beingattached to the array via an attachment site.

An aspect of the invention provides a kit for producing an array ofsingle molecules, the kit comprising: an array with attachment sites,seeds, each seed having a single attachment site, and reagents to growthe seeds into SNAPs.

An aspect of the invention provides a composition, comprising: a solidsupport; and a polymer-based molecule attached directly to the solidsupport, wherein the polymer-based molecule comprises a protein moietythat is oriented substantially opposite of the solid support, andwherein the protein moiety is accessible to affinity reagents.

An aspect of the invention provides a method of isolating biological orchemical entities on an array, the method comprising: generating aplurality of SNAPs; coupling a single biological or chemical entity toeach of the plurality of SNAPs; attaching the plurality of SNAPs to anarray, wherein the biological or chemical entity is substantiallyopposite the array, thereby isolating each biological or chemical entityof each of the plurality of SNAPs by a distance that is based on thesize of each SNAP of the plurality of SNAPs.

An aspect of the invention provides a method of separating molecules,the method comprising converting each molecule into a larger chargedmolecule.

In some cases, converting each molecule into a larger charged moleculecomprises conjugating the molecule to a biopolymer which can be grown toa desired size.

In some cases, converting each molecule into a larger charged moleculecomprises converting each molecule into a molecule 10 times larger.

In some cases, converting each molecule into a larger charged moleculecomprises conjugating each molecule to a larger charged molecule.

An aspect of the invention provides a method of producing an array ofspatially separated biological or chemical entities, the methodcomprising: obtaining a solid support with attachment sites, obtaining asample comprising a plurality of biological or chemical entities,obtaining SNAPs, each with a functional group, covalently attaching eachbiological or chemical entity of the plurality of biological or chemicalentities to a single SNAP, attaching the SNAPs to the attachment sitesof the array, thereby producing an array of spatially separatedbiological or chemical entities.

In some cases, a SNAP is a rolling circle amplification product. In somecases, a SNAP is a plasmid. In some cases, a SNAP is a DNA origamimolecule. In some cases, a SNAP is a nucleic acid cluster.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A illustrates an example method of attaching proteins to asubstrate via a structured nucleic acid particle (SNAP), wherein aprotein is covalently attached to an oligonucleotide primer which isthen annealed to a circular DNA template which contains regions ofinternal complementarity, rolling circle DNA amplification of the primeron the template results in a DNA molecule with regions of internalhybridization resulting in formation of a DNA cluster (an example of aSNAP), since the SNAP is negatively charged it can attach to apositively charged attachment site on a solid support.

FIG. 1B illustrates an alternative method of attaching proteins to asubstrate, wherein the SNAP is formed initially and then attached to aprotein.

FIG. 1C illustrates an alternative method of attaching proteins to asubstrate, wherein the SNAP is formed initially and covalently attachedto a crosslinker that is then attached to a protein.

FIG. 1D illustrates an alternative method of attaching proteins to asubstrate, wherein SNAPs are deposited onto a surface and proteins withchemical moieties react with the chemical moiety of a SNAP to attach theprotein to the SNAP.

FIG. 2 illustrates a method for producing a solid support withattachment sites arrayed at desired intervals.

FIG. 3A illustrates absorbance spectra of SNAPs produced in Example 1.

FIG. 3B illustrates fluorescence intensity of the SNAPs imaged as a dotblot.

FIG. 4 illustrates co-localization of the SNAPs and attached Deep Red200 nm beads in microscopy images of the SNAPs produced in Example 1.The Sybr Gold used to visualize the DNA fluoresces at 488 nm, and thelinked Deep Red bead fluoresces at 647 nm, regions of the images areblown up to show details, and arrow heads point to examples ofco-localization.

FIG. 5 illustrates the radii of particles in a sample comprising SNAPsbefore and after anion exchange purification.

FIG. 6 illustrates the intensities detected in fractions collectedduring the anion exchange purification of samples comprising SNAPs.

FIG. 7 illustrates the absorption spectra at 260 nm traces of differentbatches of SNAPs.

FIG. 8 illustrates the co-localization of small SNAPs on a chip.

FIG. 9 illustrates the absence of co-localization of large SNAPs on achip.

FIG. 10 illustrates the counts and number of features occupied when atitration of SNAPs is applied to a chip.

FIG. 11 illustrates the brightness of a batch of SNAPs.

FIG. 12 illustrates the measurement of the concentration of SNAPs usingan OPA assay.

FIG. 13 illustrates fluorescent images at 488 nm and 568 nm chips havingSNAPs pre- and post-conjugation with Azide-AlexaFluor 568.

FIG. 14 illustrates quantification of the intensity from fluorescentimages at 488 nm and 575 nm chips having SNAPs pre- and post-conjugationwith Azide-AlexaFluor 568.

FIG. 15 illustrates fluorescent images at 488 nm of chips having SNAPs,pre- and post-incubation with Azide-AlexaFluor 568 in excess.

FIG. 16 illustrates fluorescent images at 568 nm of chips having SNAPs,pre- and post-incubation with Azide-AlexaFluor 568 in excess.

FIG. 17 illustrates the fluorescent image of a chip having SNAPs on thesurface after click-conjugation with PE-conjugated azide.

FIGS. 18A-18F illustrate the immobilization of proteins from E. colilysate on an array having features which is coated with SNAPs. FIGS. 18Aand 18D illustrate SNAPs detected using fluorescence with a 100×100micron field of view. FIGS. 18B and 18E illustrate binding offluorescent streptavidin to biotinylated lysate (FIG. 18B), or controllysate (FIG. 18E). FIGS. 18C and 18F show colocalization of the SNAPsand streptavidin from FIGS. 18A and 18B; and FIGS. 18D and 18Erespectively.

FIGS. 19A-F illustrates the specific detection of short peptides of ashort trimer peptide epitope using SNAPs. FIGS. 19A and 19D illustrateSNAPs detected using fluorescence with a 35×35 micron field of view.FIGS. 19B and 19E illustrate fluorescence from the anti peptide aptameron the peptide treat array (FIG. 19B), or control array (FIG. 19E).FIGS. 19C and 19F show colocalization of the SNAPs and aptamer fromFIGS. 19A and 19B; and FIGS. 19D and 19E respectively.

DETAILED DESCRIPTION OF THE INVENTION

Microarrays and nanoarrays having a plurality of molecules spatiallydistributed over and stably associated with the surface of a solidsupport are becoming an increasingly important tool in bioanalysis andrelated fields. Microarrays of both polypeptides and polynucleotideshave been developed and find use in a variety of applications, such asgene sequencing, monitoring gene expression, gene mapping, bacterialidentification, drug discovery, and combinatorial chemistry. One area inparticular in which microarrays find use is in gene expression analysis.

In some instances it may be desirable to produce a microarray ornanoarray wherein a plurality of biological or chemical entities arespatially distributed over and stably associated with the surface of asolid support such that each individual biological or chemical entity isspatially separated from each other biological or chemical entity.

In some embodiments this disclosure provides methods of producing anarray of spatially separated biological or chemical entities, a methodmay comprise: obtaining a solid support with attachment sites, obtaininga sample comprising biological or chemical entities, obtaining seeds,each with a functional group, covalently attaching each biological orchemical entity to a single seed via the functional group, growing eachattached seed to a SNAP (Structured Nucleic Acid Particles) of desiredsize, attaching the SNAPs to the attachment sites of the array, therebyproducing a regular array of biological or chemical entities. In someinstances, SNAPs can be any type of DNA based nanoparticle, such asrolling circle amplification based nanoparticles, plasmids, or DNAorigami nanoparticles.

Examples of such methods of producing an array of entities such asproteins are provided in FIGS. 1 A-D. FIG. 1A depicts a method whichbegins with the attachment of a protein to an oligonucleotide primer viaa linker. The primer can be then annealed to a circular DNA template,and rolling circle amplification can be performed to produce a SNAP(indicated in this example as a DNA cluster). The SNAP can be thendeposited onto a chip. In this example, the negative charge of the DNAbackbone can interact with positively charged features of an array, suchthat the SNAP becomes immobilized on the array.

FIG. 1B depicts a method which begins with a primer having a linkerinitiating rolling circle amplification with a circular DNA template.The resulting SNAP (indicated in this example as a DNA cluster) thuscomprises a linker, which can then be conjugated to a protein. The SNAPcan be then deposited onto a chip. In this example, the negative chargeof the DNA backbone can interact with positively charged features of anarray, such that the SNAP becomes immobilized on the array.

FIG. 1C depicts a method which begins with a primer initiating rollingcircle amplification with a circular DNA template. The resulting SNAP(indicated in this example as a DNA cluster) can then be joined with acrosslinker, which can then be conjugated with a protein, to result in aSNAP which is crosslinked to a protein. The SNAP can be then depositedonto a chip. In this example, the negative charge of the DNA backbonecan interact with positively charged features of an array, such that theSNAP becomes immobilized on the array.

FIG. 1D depicts SNAPs which have already been created, for example byrolling circle amplification or other acceptable method. These SNAPs canbe then deposited onto a chip. For example, the negative charge of theDNA backbone can interact with positively charged features of an array,such that the SNAP becomes immobilized on the array. Separately,proteins can be modified with chemical handles which can bind a chemicalmoiety which can be on the SNAPs. The handled proteins can then beapplied to the SNAPs, such that they covalently attach to the SNAPs.

In some embodiments this disclosure provides arrays of single moleculesand methods and kits for producing arrays of single molecules. In someembodiments this disclosure provides arrays of biological or chemicalentities and methods and kits for producing arrays of biological orchemical entities. In some examples, an array of biological or chemicalentities may comprise an ordered series of biological or chemicalentities arrayed on a solid support. In other examples, an array ofbiological or chemical entities may comprise an irregular array ofbiological or chemical entities.

In some examples, biological or chemical entities on an array may beseparated by less than 10 nm, about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm,60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1 m, 1.2 m, 1.4 m,1.6 m, 1.8 m, 2 m, 2.5 m, 3 m, 4 m, 5 m, 6 m, 7 m, 8 m, 9 m, 10 m, 11 m,12 m, 13 m, 14 m, 15 m, 16 m, 17 m, 18 m, 19 m, 20 m, 25 m, 30 m, 40 m,50 m, 75 m, 100 m, 200 m, 300 m, 400 m, 500 m, or more than 500 m. Insome cases, biological or chemical entities on the array may beseparated by between about 50 nm and about 1 m, about 50 nm and about500 nm, about 100 nm and about 400 nm, about 200 nm and about 300 nm,about 500 nm and about 10 m, about 50 nm and about 1 m, or about 300 nmand about 1 m. In some cases, the spacing of biological or chemicalentities on the array may be determined by the presence of attachmentsites arrayed on a solid support

In some embodiments an array is created on a solid support. The solidsupport may be any solid surface to which molecules can be covalently ornon-covalently attached. Non-limiting examples of solid substratesinclude slides, surfaces of elements of devices, membranes, flow cells,wells, chambers, and macrofluidic chambers. Solid supports used hereinmay be flat or curved, or can have other shapes, and can be smooth ortextured. In some cases, solid support surfaces may contain microwells.In some cases, substrate surfaces may contain nanowells. In some cases,solid support surfaces may contain one or more microwells in combinationwith one or more nanowells. In some embodiments, the solid support canbe composed of glass, carbohydrates such as dextrans, plastics such aspolystyrene or polypropylene, polyacrylamide, latex, silicon, metalssuch as gold, chromium, titanium, or tin, titanium oxide, tin oxide, orcellulose. In some examples, the solid support may be a slide or a flowcell.

In some embodiments, surfaces of the solid support may be modified toallow or enhance covalent or non-covalent attachment of molecules suchas the SNAPs described herein. The solid support and process formolecule attachment are preferably stable for repeated binding, washing,imaging and eluting steps. In some cases, surfaces may be modified tohave a positive or negative charge. In some cases, surfaces may befunctionalized by modification with specific functional groups, such asmaleic or succinic moieties, or derivatized by modification with achemically reactive group, such as amino, thiol, or acrylate groups,such as by silanization. Suitable silane reagents includeaminopropyltrimethoxysilane, aminopropyltriethoxysilane and4-aminobutyltriethoxysilane. The surfaces may be functionalized withN-Hydroxysuccinimide (NHS) functional groups. Glass surfaces can also bederivatized with other reactive groups, such as acrylate or epoxy,using, e.g., epoxysilane, acrylatesilane or acrylamidesilane.

In some embodiments, the solid support may be modified to reducenon-specific attachment of SNAPs to the solid support. In someembodiments, the solid support may be modified to reduce non-specificattachment of biological entities and/or chemical entities to the solidsupport. In some embodiments, the solid support may be passivated. Insome further embodiments, the surface of the solid support may bepassivated. In some embodiments, the passivation layer may includediamond-like carbon, hexa-methyldisilizane, Teflon, fluorocarbon, apolymer such as polyethylene glycol (PEG) and/or Parylene. In someembodiments, a solid support may be passivated by the attachment ofPolyethylene glycol (PEG) molecules across the solid support. In someembodiments, a solid support may be passivated using salmon sperm DNA,glycols, albumin, or a combination of the above. In some embodiments, asolid support may be passivated using one or more components selectedfrom the group consisting of salmon sperm DNA, glycols, and albumin. Insome embodiments, passivation components may be exposed to a surface. Insome embodiments, passivation components may not be covalently bound toa surface. In some embodiments, passivation materials may be notcovalently bound to the solid support.

In some embodiments, the solid support may be modified across the entiresurface to which molecules are to be attached. In other embodiments, thesolid support may contain regions which are modified to allow attachmentof molecules and regions which are not modified, or regions which aremodified to decrease attachment of molecules and regions which are notmodified, or regions which are modified to increase attachment ofmolecules and regions which are modified to decrease attachment ofmolecules. In some cases attachment sites may be created in an array,for example an ordered array.

An ordered array of attachment sites may be created by, for example,photolithography, Dip-Pen nanolithography, nanoimprint lithography,nanosphere lithography, cluster lithography, nanopillar arrays, nanowirelithography, scanning probe lithography, thermochemical lithography,thermal scanning probe lithography, local oxidation nanolithography,molecular self-assembly, stencil lithography, or electron-beamlithography. Attachment sites in an ordered array may be located suchthat each attachment site is less than 20 nanometers (nm), or about 20nm, about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm,about 175 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm,about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm,about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm,about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm,about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm,about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm,about 925 nm, about 950 nm, about 975 nm, about 1000 nm, about 1025 nm,about 1050 nm, about 1075 nm, about 1100 nm, about 1125 nm, about 1150nm, about 1175 nm, about 1200 nm, about 1225 nm, about 1250 nm, about1275 nm, about 1300 nm, about 1325 nm, about 1350 nm, about 1375 nm,about 1400 nm, about 1425 nm, about 1450 nm, about 1475 nm, about 1500nm, about 1525 nm, about 1550 nm, about 1575 nm, about 1600 nm, about1625 nm, about 1650 nm, about 1675 nm, about 1700 nm, about 1725 nm,about 1750 nm, about 1775 nm, about 1800 nm, about 1825 nm, about 1850nm, about 1875 nm, about 1900 nm, about 1925 nm, about 1950 nm, about1975 nm, about 2000 nm, or more than 2000 nm from any other attachmentsite.

In some cases, the spacing of attachment sites on the solid support maybe selected depending on the size of the SNAPs to be used. For examplethe spacing of the attachment sites may be selected such that thedistance between the edges of any two attachment sites is greater thanthe diameter of the SNAP used.

In some cases, the size of the attachment sites on the solid support maybe selected depending on the size of the SNAPs to be used. For examplethe size of the attachment sites may be selected such that the diameterof each attachment sites is less than the diameter of the SNAP used.

In some cases, the attachment sites may be provided in microwells ornanowells.

In some cases, functional groups may be present in a random spacing andmay be provided at a concentration such that functional groups are onaverage at least about 50 nm, about 100 nm, about 150 nm, about 200 nm,about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm,about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm,about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm,about 1000 nm, or more than 100 nm from any other functional group.

The solid support may be indirectly functionalized. For example, thesolid support may be PEGylated and a functional group may be applied toall or a subset of the PEG molecules.

In some cases, the efficiency of attachment of the SNAPs to the solidsupport may be high. In some cases, the efficiency of attachment of theSNAPs to the solid support may be moderate. In some cases, theefficiency of attachment of the SNAPs to the solid support may be low.The efficiency of the attachment of the SNAPs to the solid support maybe influenced by many factors, including, but not limited to: sequenceof clusters, size of SNAPs relative to size of a corresponding bindingpatch, the extent to which SNAPs have had their structure modified insuch a way so as to influence their binding, age of SNAPs, storageconditions of a buffer or buffers that come into contact with SNAPs,storage conditions of SNAPs, pH or other properties of solvent in whichthe binding is hoping to be achieved can massively affect, percentagesof positive cations, and temperature. In some cases, the reliability ofattachment of the SNAPs to the solid support may be high. In some cases,the reliability of attachment of the SNAPs to the solid support may bemoderate. In some cases, the reliability of attachment of the SNAPs tothe solid support may be low.

In some embodiments the solid support may be optically opaque. In somecases the solid support may be optically clear at one or morewavelengths. In some cases, the solid support may be partially opticallyclear, or may be optically clear in some regions. For example a solidsupport may be optically opaque in regions that are not functionalized,and optically clear in regions that are functionalized.

FIG. 2 illustrates a method for producing a solid support withattachment sites arrayed at desired intervals. Initially, a substrate isprovided. In some embodiments, the substrate may be glass. Inparticular, in some embodiments, the substrate may be amorphous glass,fused silica, or quartz, among other examples. In some embodiments, thesubstrate may be silicon. In some embodiments, the thickness of thesubstrate may be less than 100 microns, 100 microns, 150 microns, 200microns, 300 microns, 400 microns, 500 microns, 600 microns, 700microns, 800 microns, 900 microns, 1 millimeter, 2 millimeters, or morethan 2 millimeters.

Initially, the substrate is cleaned, such as with a piranha cleaning. Insome embodiments, a substrate may be cleaned using a strong acid so asto clean the substrate without etching the substrate. In someembodiments, the substrate may be cleaned using a detergent.Alternatively, the substrate may be cleaned with solvent, sonication orwith plasma such as O₂ or N₂ plasma, or with a combination thereof.

Once the substrate has been cleaned, a chrome layer is deposited on thebackside of the substrate. Deposition methods may include, for example,evaporation or sputtering. In some embodiments, a backside chromeevaporation may not be applied when a substrate is opaque. A backsidechrome evaporation may have a thickness of one Angstrom, two Angstroms,10 Angstroms, 10 nanometers, 20 nanometers, 30 nanometers, 40nanometers, 50 nanometers, 60 nanometers, 70 nanometers, 80 nanometers,90 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250nanometers, 300 nanometers, 400 nanometers, 500 nanometers, or more than500 nanometers. Alternatively, other metals can be used for depositionon the backside of the substrate, such as Aluminum, Tungsten, and/orTitanium, among other examples. Alternatively, dielectric mirrors can beused for deposition on the backside of the substrate.

Further, fiducials may be created on the front side of the substrate.Fiducials may be created by adding at least one layer of material and bypatterning this at least one layer. In some embodiments, such materialcan be chrome, and/or such materials may be other metals like tungstenor gold. Alternatively, dielectric mirrors could be used as a materialfor fiducials. Alternatively, metal oxide could be used for thefiducials as for example ZrO₂. The patterning of such materials can beperformed in a variety of ways. A first way to pattern the fiducialmaterial is to deposit a blanket layer of the material, then to protectthis material in selected areas and remove the material in the areaswhere it is not protected. This can for example be achieved by coatingthe front side of the substrate with photosensitive material (e.g.photoresist), patterning this photoresist by exposing it to UV lightthrough a mask and then developing it. The etching of the fiducialmaterial can then be performed by wet etch (for example acid) or dryetch (for example Reactive Ion Etching, RIE). Alternatively, thephotoresist may be deposited and patterned first. In some embodimentswhere the photoresist is deposited and patterned first, areas aredefined that are free of such photoresist and then the fiducial materialmay be deposited on top of the photoresist. The photoresist may then beremoved (for example, in a solvent bath with sonication) and thefiducial material may be left on the areas that were initially free ofphotoresist (e.g., using a lift-off technique). Alternatively, fiducialsmay be created by removing material from the substrate in selectedareas, for example by patterning a layer of photoresist on the frontside of the substrate and then by dry etching the substrate in the areasthat are not coated with photoresist. In an another alternative,fiducials may be defined by modifying the substrate locally (for exampleby laser melting and/or fractioning). Fiducials may come in a variety ofshapes, lines, and/or orientations. In some embodiments, a pattern offiducials may be applied to the substrate. In yet another embodiment,the shape of fiducials may vary in order to code information about theirlocation on the surface of the substrate.

Once a pattern of fiducials is created on the front side of thesubstrate, this front side may be differentially coated to definefeatures where the biological objects of interest (for example, nucleicacid clusters covalently attached to a protein) may be immobilized. In afirst embodiment, the surface may be differentially patterned with twosilanes, for example HMDS or a PEG-silane in the field and APTES on theimmobilization spots. This differential patterning is achieved by, forexample, depositing an initial HMDS layer on the surface, followed by alift-off layer, followed by an optional anti-reflective layer, andfollowed by a photoresist layer. In some embodiments, an anti-reflectivelayer may not be provided when an opaque substrate is being used.

Once the photoresist is applied, a second lithography step may beprovided. In particular, desired features may be provided. In someembodiments, desired features may have a length of approximately 300 nm.In some embodiments, features may have a length of less than 50 nm, 100nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600nm, 700 nm, or more than 700 nm. In some further embodiments, one ormore layers deposited on the surface to perform this second lithographymay not be etched by the developing step of this second lithography (forexample, the antireflective coating).

In embodiments where a backside coating is provided, the backsidecoating may be removed, such as through the use of a wet etch or dryetch etc. Further, a directional reactive ion etch (RIE) may be providedso as to remove layers that haven't been removed by the lithography step(for example the antireflective coating).

Once the holes have been provided, cleaning may be performed. As seen inFIG. 2, an oxygen plasma cleaning and activation step is provided. Oncethe chip has been cleaned, an amino-silane deposition may be provided.Once the amino-silane deposition is provided, portions of the chipmanufacture may be lifted-off, such as using hot DMF. Further, asonication step may be performed. The resulting chip may be used in flowcells for assessments of biological assays.

In an alternative embodiment, the surface may be differentiallypatterned with a silane layer and a metal layer (for example,(3-Aminopropyl)triethoxysilane (APTES) on the immobilization spots andchrome in the field). In another embodiment, the surface may bedifferentially patterned with a silane layer and a metal oxide layer(for example a PEG-silane layer in the field and a ZrO₂ layer on theimmobilization spots). In yet another embodiment, the surface may bedifferentially patterned with a silane layer on the immobilization spots(for example, acyl protein thioesterases (APTS)) and a metal oxide layer(for example a ZrO₂) and a PEG-phosphonic acid layer in the field.

The biological or chemical entities of this disclosure may be anybiological or chemical entities for which spatial separation is desired.In some embodiments, the biological or chemical entities are proteins.In some cases, the proteins may be proteins from a cell or tissuehomogenate, from a biological fluid, or from an environmental sample. Insome cases, the biological or chemical entities may be antibodies. Insome embodiments the biological or chemical entities are nucleic acids.For example the biological or chemical entities may be DNAs, RNAs,mRNAs, tRNAs, or miRNAs. In some embodiments the biological or chemicalentities are carbohydrates. In some embodiments, the biological orchemical entities are complex polymers. In some embodiments thebiological or chemical entities are small molecules, for examplechemical compounds rather than complex polymers.

The biological or chemical entities of this disclosure may be attachedto seeds. These seeds are molecules which can be used as a starting‘seed’ to grow a larger polymeric molecule. The seed may be a monomercapable of being grown into a polymer, or may comprise a monomer capableof being grown into a polymer. Generally, the seeds are molecules whichcan be covalently attached to the molecules. The seeds may have apolarity such that only one functional group of the seed is able to bindto a molecule of the molecules to be separated, while another one ormore functional groups of the seed can form the starting point for apolymer.

Examples of monomers which may be present in the seeds include, but arenot limited to, oligonucleotides, carbohydrates, proteins, amyloids,fibrils, and tetratricopeptide repeats. In some cases the seeds aresmall molecules.

The seeds may comprise a monomer and a functional group able to bind toa biological or chemical entity to be separated. Examples of suchfunctional groups may include, but are not limited to, amines, thiols,carboxylic acids, triple bonds, double bonds, epoxides, alkynes,alkenes, cycloalkynes, azides, cyclo-octynes, cycloalkynes, norbornenes,tetrazines, cyclloctanes, epoxides, and hydroxyls. In some cases, theseed may comprise a functional group that is compatible with a clickchemistry. In some cases, the seed may also comprise a linker or spacerbetween the seed and the functional group. In some cases, the linker orspacer may comprise a photo-cleavable bond. In some cases, the seed maycomprise an oligonucleotide conjugated to an amine group on the 5′terminal. In some cases, the seed may comprise an oligonucleotideconjugated to a click chemistry component on the 5′ terminal.

In some cases, bioconjugation may be used to form a covalent bondbetween two molecules, at least one of which is a biomolecule.Bioconjugation may be formed but not limited to via chemicalconjugation, enzymatic conjugation, photo-conjugation,thermal-conjugation, or a combination thereof. (Spicer, C. D., Pashuck,E. T., & Stevens, M. M., Achieving Controlled Biomolecule-BiomaterialConjugation. Chemical Reviews., 2018, 118, Pgs. 7702-7743, and Greg T.Hermanson, “Bioconjugate Techniques”, Academic Press; 3^(rd) Edition,2013, herein incorporated by reference for this disclosure). In somecases, both the seed and the biological (e.g. SNAP) or chemical entitymay be functionalized. Functionalizing both partners may improve theefficiency or speed of a conjugation reaction. For example, a sulfhydrylgroup (—SH) or amine (—NH₂) of a chemically active site of a seed,biological, or chemical entity may be functionalized to allow forgreater reactivity or efficiency of a conjugation reaction. Any of avariety of sulfhydryl-reactive (or thiol-reactive) or amine conjugationchemistries may be used to couple chemical moieties to sulfhydryl oramine groups. Examples include, but are not limited to, use ofhaloacetyls, maleimides, aziridines, acryloyls, arylating agents,vinylsulfones, pyridyl disulfides, TNB-thiols and/or othersulfhydryl-reactive/amine-reactive/thiol-reactive agents. Many of thesegroups conjugate to sulfhydryl groups through either alkylation (e.g.,by formation of a thioether or amine bond) or disulfide exchange (e.g.,by formation of a disulfide bond). More strategies and detail regardingreactions for bioconjugation are described down below and may beextended to other appropriate biomolecules.

Bioconjugation can be accomplished in part by a chemical reaction of achemical moiety or linker molecule with a chemically active site on thebiomolecule. The chemical conjugation may proceed via an amide formationreaction, reductive amination reaction, N-terminal modification, thiolMichael addition reaction, disulfide formation reaction,copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction,strain-promoted alkyne-azide cycloaddtion reaction (SPAAC),Strain-promoted alkyne-nitrone cycloaddition (SPANC), inverselectron-demand Diels-Alder (IEDDA) reaction, oxime/hydrazone formationreaction, free-radical polymerization reaction, or a combinationthereof. Enzyme-mediated conjugation may proceed via transglutaminases,peroxidases, sortase, SpyTag-SpyCatcher, or a combination thereof.Photoconjugated and activation may proceed via photoacrylatecross-linking reaction, photo thiol-ene reaction, photo thiol-ynereaction, or a combination thereof. In some cases, conjugation mayproceed via noncovalent interactions, these may be throughself-assembling peptides, binding sequences, host-guest chemistry,nucleic acids, or a combination thereof.

In some cases, site-selectivity methods may be employed to modifyreaction moieties of biomolecules to increase conjugation efficiency,ease of use, reproducibility. Three common strategies are typicallyemployed for site-selective bioconjugation (i) Modification strategiesthat can select a single motif among many, rather than targeting ageneric reactive handle. This may be determined by surrounding asequence, local environment, or subtle differences in reactivity. Theability of enzymes to modify a specific amino acid within a proteinsequence or a glycan at a single position are particularly prominent.Reactions that display exquisite chemo-selectivity also fall within thiscategory, such as those that target the unique reactivity of the proteinN-terminus or the anomeric position of glycans. (ii) The site-specificincorporation of unnatural functionalities, by hijacking nativebiosynthetic pathways may be utilized. (iii) The installation of uniquereactivity via chemical synthesis may be utilized. The complete orpartial synthesis of peptides and oligonucleotides is widespread,particularly using solid-phase approaches. These techniques allow accessto sequences of up to 100 amino acids or 200 nucleotides, with theability to install a wide variety of functionalized monomers withprecise positional control.

In some cases, chemical conjugation techniques may be applied forcreating biomaterial-biomolecule conjugates. Functional groups used forbioconjugation may be native to the biomolecule or may be incorporatedsynthetically. In the illustrations below, R and R′ may be a biomolecule(for example, but not limited to: SNAP, proteins, nucleic acids,carbohydrates, lipids, metabolites, small molecules, monomers,oligomers, polymers) and/or a solid support.

In some cases, reductive amination may be utilized for bioconjugation.Amines can react reversibly with aldehydes to form a transient iminemoiety, with accompanying elimination of water. This reaction takesplace in rapid equilibrium, with the unconjugated starting materialsbeing strongly favored in aqueous conditions due to the highconcentration of water. However, in a second step the unstable imine canbe irreversibly reduced to the corresponding amine via treatment withsodium cyanoborohydride. This mild reducing reagent enables theselective reduction of imines even in the presence of unreactedaldehydes. As a result, irreversible conjugation of a biomolecule cangradually occur to a biomaterial of interest. In contrast, strongerreducing agents such as sodium borohydride are also able to reducealdehydes. This two-step reductive amination process can also beutilized for the modification of ketones. For example, reductiveamination has therefore been primarily used for the modification ofsodium periodate-treated alginate and chitosan scaffolds. The order ofreactivity may also be reversed for the attachment of reducing sugars,by exploiting the terminal aldehyde/ketone generated in the open-chainform. This strategy, for example, may be exploited to mimic theglucosylation and galactosylation patterns of native collagen in ECM,via reductive amination of maltose and lactose respectively.

In some cases, isothiocyanates of a biomolecule or solid support may beutilized for bioconjugation. For example, isothiocyanate of abiomolecule may react with nucleophiles such as amines, sulfhydryls, thephenolate ion of tyrosine side chains or other biomolecules to form astable bond between two molecules.

In some cases, an isocyanate of a biomolecule or solid support may beutilized for bioconjugation. For example, isocyanates can react withamine-containing molecules to form stable isourea linkages.

In some cases, an acyl azide of a biomolecule or solid support may beutilized for bioconjugation. For example, acyl azide are activatedcarboxylate groups that can react with primary amines to form amidebonds.

In some cases, an amide of a biomolecule or solid support may beutilized for bioconjugation. For example, the use of reactiveN-hydroxysuccinimide (NHS) esters is particularly widespread. WhileNHS-esters can be preformed, often they are instead generated in situthrough the use of N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide(EDC) coupling chemistry and coupled directly to the species ofinterest. Although formation of the activated NHS-ester is favored undermildly acidic conditions (pH˜5), subsequent amide coupling isaccelerated at higher pHs at which the amine coupling partner is notprotonated. One-step modification at an intermediate pH of ˜6.5 ispossible. Conjugation is typically undertaken by first forming theactive NHS-ester at pH 5, before raising the pH to ˜8 and adding theamine coupling partner in a two-step procedure. In some cases,water-soluble derivative sulfo-NHS may be utilized as an alternative. Insome cases, NHS esters of a biomolecule can react and couple withtyrosine, serine, and threonine —OH groups as opposed to N-terminalα-amines and lysine side-chain ε-amines.

In some cases, a sulfonyl chloride of a biomolecule or solid support maybe utilized for bioconjugation. For example, reaction of a sulfonylchloride compound with a primary amine-containing molecule proceeds withloss of the chlorine atom and formation of a sulfonamide linkage.

In some cases, a tosylate ester of a biomolecule or solid support may beutilized for bioconjugation. For example, reactive groups comprisingtosylate esters can be formed from the reaction of 4-toluenesulfonylchloride (also called tosyl chloride or TsCl) with a hydroxyl group toyield the sulfonyl ester derivative. The sulfonyl ester may couple withnucleophiles to produce a covalent bond and may result in a secondaryamine linkage with primary amines, a thioether linkage with sulf-hydrylgroups, or an ether bond with hydroxyls.

In some cases, a carbonyl of a biomolecule or solid support may beutilized for bioconjugation. For example, carbonyl groups such asaldehydes, ketones, and glyoxals can react with amines to form Schiffbase intermediates which are in equilibrium with their free forms. Insome cases, the addition of sodium borohydride or sodiumcyanoborohydride to a reaction medium containing an aldehyde compoundand an amine-containing molecule will result in reduction of the Schiffbase intermediate and covalent bond formation, creating a secondaryamine linkage between the two molecules.

In some cases, an epoxide or oxirane of a biomolecule or solid supportmay be utilized for bioconjugation. For example, an epoxide or oxiranegroup of a biomolecule may react with nucleophiles in a ring-openingprocess. The reaction can take place with primary amines, sulfhydryls,or hydroxyl groups to create secondary amine, thioether, or ether bonds,respectively.

In some cases, a carbonate of a biomolecule or solid support may beutilized for bioconjugation. For example, carbonates may react withnucleophiles to form carbamate linkages, disuccinimidyl carbonate, canbe used to activate hydroxyl-containing molecules to form amine-reactivesuccinimidyl carbonate intermediates. In some cases, this carbonateactivation procedure can be used in coupling polyethylene glycol (PEG)to proteins and other amine-containing molecules. In some cases,nucleophiles, such as the primary amino groups of proteins, can reactwith the succinimidyl carbonate functional groups to give stablecarbamate (aliphatic urethane) bonds

In some cases, an aryl halide of a biomolecule or solid support may beutilized for bioconjugation. For example, aryl halide compounds such asfluorobenzene derivatives can be used to form covalent bonds withamine-containing molecules like proteins. Other nucleophiles such asthiol, imidazolyl, and phenolate groups of amino acid side chains canalso react to form stable bonds with a biomolecule or solid support. Insome cases, fluorobenzene-type compounds have been used as functionalgroups in homobifunctional crosslinking agents. For example, theirreaction with amines involves nucleophilic displacement of the fluorineatom with the amine derivative, creating a substituted aryl amine bond.

In some cases, an imidoester of a biomolecule or solid support may beutilized for bioconjugation. For example, the α-amines and ε-amines ofproteins may be targeted and crosslinked by reacting withhomobifunctional imidoesters. In some cases, after conjugating twoproteins with a bifunctional imidoester crosslinker, excess imidoesterfunctional groups may be blocked with ethanolamine.

In some cases carbodiimides may be utilized for bioconjugation.Generally, carbodiimides are zero-length crosslinking agents that may beused to mediate the formation of an amide or phos-phoramidate linkagebetween a carboxylate group and an amine or a phosphate and an amine,respectively. Carbodiimides are zero-length reagents because in formingthese bonds no additional chemical structure is introduced between theconjugating molecules. In some cases, N-substituted carbodiimides canreact with carboxylic acids to form highly reactive, O-acylisoureaderivatives. This active species may then react with a nucleophile suchas a primary amine to form an amide bond. In some cases, sulfhydrylgroups may attack the active species and form thioester linkages. Insome cases, hydrazide-containing compounds can also be coupled tocarboxylate groups using a carbodiimide-mediated reaction. Usingbifunctional hydrazide reagents, carboxylates may be modified to possessterminal hydra-zide groups able to conjugate with other carbonylcompounds.

In some cases, a biomolecule containing phosphate groups, such as the5′phosphate of oligonucleotides, may also be conjugated toamine-containing molecules by using a carbodiimide-mediated reaction.For example, the carbodiimide of a biomolecule may activate thephosphate to an intermediate phosphate ester similar to its reactionwith carboxylates. In the presence of an amine, the ester reacts to forma stable phosphoramidate bond.

In some cases, an acid anhydride of a biomolecule or solid support maybe utilized for bioconjugation. Anhydrides are highly reactive towardnucleophiles and are able to acylate a number of the importantfunctional groups of proteins and other biomolecules. For example,protein functional groups able to react with anhydrides include but notlimited to the ε-amines at the N-terminals, the ε-amine of lysine sidechains, cysteine sulfhydryl groups, the phenolate ion of tyrosineresidues, and the imid-azolyl ring of histidines. In some cases, thesite of reactivity for anhydrides in protein molecules is modificationof any attached carbohydrate chains. In some cases, in addition to aminogroup modification in a polypeptide chain, glycoproteins may be modifiedat their polysaccharide hydroxyl groups to form esterified derivatives.

In some cases, a fluorophenyl ester of a biomolecule or solid supportmay be utilized for bioconjugation. Fluorphenyl esters can be anothertype of carboxylic acid derivative that may react with amines consistsof the ester of a fluorophenol compound, which creates a group capableof forming amide bonds with proteins and other molecules. In some cases,fluorophenyl esters may be: a pentafluorophenyl (PFP) ester, atetrafluorophenyl (TFP) ester, or a sulfo-tetrafluoro-phenyl (STP)ester. In some cases, fluorophenyl esters react with amine-containingmolecules at slightly alkaline pH values to give the same amide bondlinkages as NHS esters.

In some cases, hydroxymethyl phosphine of a biomolecule or solid supportmay be utilized for bioconjugation. Phosphine derivatives withhydroxymethyl group substitutions may act as bioconjugation agents forcoupling or crosslinking purposes. For example, tris(hydroxymethyl)phosphine (THP) and β-[tris(hydroxymethyl)phos-phino] propionic acid(THPP) are small trifunctional compounds that spontaneously react withnucleophiles, such as amines, to form covalent linkages.

In some cases, the thiol reactivity of a biomolecule or solid supportmay be utilized for bioconjugation. For example, the thiol group ofcysteine is the most nucleophilic functional group found among the 20proteinogenic amino acids. Through careful control of pH, selectivemodification over other nucleophilic residues such as lysine can bereadily achieved. Another example, thiol modification ofoligonucleotides may be used to enable derivatization, though the easewith which alternative reactive handles with enhanced chemicalorthogonality can be installed has limited use forbiomaterial-conjugation. Further, the conjugate addition of thiols toα,β-unsaturated carbonyls, also known as Michael addition, may be usedto form polypeptide conjugates in the fields of tissue engineering,functional materials, and protein modification. In general, reactionrates and conjugation efficiencies are primarily controlled by threefactors and may be modified as needed: (i) the pK_(a) of the thiol; (ii)the electrophilicity of the Michael-acceptor; (iii) the choice ofcatalyst. Regarding (i): the thiolate anion is the active nucleophileduring Michael addition, and the propensity of the thiol to undergodeprotonation may determine thiolate concentration and thus reactionrates. For example, the lower pK_(a) of aromatic thiols, when comparedto their aliphatic counterparts, leads to a higher rate of reaction ratea weak base is used to catalyze the. As a result, local structure cansignificantly alter conjugation efficiency, particularly for polypeptidesubstrates. The pK_(a) and reactivity of cysteine containing peptidescan be altered significantly through rational choice of surroundingamino acids, the presence of positively charged amino acids, such aslysine and arginine, acts to lower the thiol pK_(a) and thus enhancereactivity. Regarding (ii): the Michael-acceptor becomes more electrondeficient it becomes more activated toward nucleophilic attack, and thusreaction rates increase. Within the most widely utilized acceptors inthe biomaterial field, a trend of reactivity can be generalized asmaleimides>vinyl sulfones>acrylates>acrylamides>methacrylates. Regarding(iii) Michael additions can be accelerated by either basic ornucleophilic catalysis (although both act by increasing theconcentration of the active thiolate).

In some cases, the unique nucleophilicity of thiols can be exploited forselective reaction with a number of alternative electrophiles, whichallow efficient and selective biomolecule attachment to be achieved. Forexample, one such group are α-halocarbonyls, with iodoacetamide basedreagents finding particular utility. Higher thiol selectivity may beachieved using less electrophilic bromo and even chloro derivatives,though reactivity is also drastically reduced. More recently,methylsulfonyl heteroaromatic derivatives have emerged as promisingreagents for thiol-specific conjugation. In other cases, alternativethiol-reactive handles, such as disulfide-bridging pyridazinediones,carbonylacrylic reagents, and cyclopropenyl ketones may be utilized forbioconjugation.

In some cases, sulfhydryl of a biomolecule or solid support may beutilized for bioconjugation. In some cases, three forms of activatedhalogen derivatives can be used to create sulfhydryl-reactive compounds:haloacetyl, benzyl halides, and alkyl halides. In each of thesecompounds, the halogen group may be easily displaced by an attackingnucleophilic substance to form an alkylated derivative with loss of HX(where X is the halogen and the hydrogen comes from the nucleophile).Haloacetyl compounds and benzyl halides typically are iodine or brominederivatives, whereas the halo-mustards mainly employ chlorine andbromine forms. Iodoacetyl groups have also been used successfully tocouple affinity ligands to chromatography supports.

In some cases, a maleimide of a biomolecule or solid support may beutilized for bioconjugation. The double bond of maleimides may undergoan alkylation reaction with sulfhydryl groups to form stable thioetherbonds.

In some cases, an aziridine of a biomolecule or solid support may beutilized for bioconjugation. The highly hindered nature of thisheterocyclic ring gives it strong reactivity toward nucleophiles. Forexample, sulfhydryls will react with aziridine-containing reagents in aring-opening process, forming thioether bonds. The simplest aziridinecompound, ethylenimine, can be used to transform available sulfhydrylgroups into amines. In some cases, substituted aziridines may be used toform homobifunctional and trifunctional crosslinking agents.

In some cases, thiol-maleimide reactions are particularly useful forundertaking conjugation at low concentrations or when requiringextremely high efficiencies due to the value of the biomoleculesubstrate. The use of maleimides in bioconjugation is further enhancedby the ease with which they may be introduced into a wide range ofscaffold materials, through the modification of amines with thedifunctional reagent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, more commonly referred to by its abbreviationSMCC. For example, this reagent has been widely used to first introducea maleimide reactive handle on a biomaterial of choice and then toenable the attachment of both peptides and growth factors to producebioactive scaffolds.

In some cases, an acryloyl of a biomolecule or solid support may beutilized for bioconjugation. The reactive double bonds are capable ofundergoing additional reactions with sulfhydryl groups. In some cases,the reaction of an acryloyl compound with a sulfhydryl group occurs withthe creation of a stable thioether bond. In some cases, the acryloyl hasfound use in the design of the sulfhydryl-reactive fluorescent probe,6-acryloyl-2-dimethylaminonaphthalene.

In some cases, an aryl group of a biomolecule or solid support may beutilized for bioconjugation with a sulfhydryl group. Although arylhalides are commonly used to modify amine-containing molecules to formaryl amine derivatives, they also may react quite readily withsulfhydryl groups. For example, fluorobenzene-type compounds have beenused as functional groups in homobifunctional crosslinking agents. Theirreaction with nucleophiles involves bimolecular nucleophilicsubstitution, causing the replacement of the fluorine atom with thesulfhydryl derivative and creating a substituted aryl bond. Conjugatesformed with sulfhydryl groups are reversible by cleaving with an excessof thiol (such as DTT).

In some cases, the disulfide group of a biomolecule or solid support maybe utilized for bioconjugation. In some cases, compounds containing adisulfide group are able to participate in disulfide exchange reactionswith another thiol. The disulfide exchange (also called interchange)process involves attack of the thiol at the disulfide, breaking the—S—S— bond, with subsequent formation of a new mixed disulfidecomprising a portion of the original disulfide compound. The reductionof disulfide groups to sulfhydryls in proteins using thiol-containingreductants proceeds through the intermediate formation of a mixeddisulfide. In some cases, crosslinking or modification reactions may usedisulfide exchange processes to form disulfide linkages withsulfhydryl-containing molecules.

In some cases, disulfide bonds may be utilized for bioconjugation. Forexample, the use of disulfide exchange reactions may be favored forintroducing peptides or proteins of interest. The most commonly usedreagents in tissue engineering are based upon reactivepyridylthio-disulfides, which undergo rapid thiol-exchange to releasethe poorly nucleophilic and spectroscopically active 2-mercaptopyridine.Additionally, due to the reversible nature of disulfide bond formation,cleavage can be controlled with temporal precision by the addition ofreducing agents such as dithiothreitol (DTT) or glutathione.

In some cases, a pyridyl dithiol functional group may be used in theconstruction of crosslinkers or modification reagents forbioconjugation. Pyridyl disulfides may be created from available primaryamines on molecules through the reaction of 2-iminothiolane in tandemwith 4,4′-dipyridyl disulfide. For instance, the simultaneous reactionamong a protein or other biomolecule, 2-iminothiolane, and4,4′-dipyri-dyl disulfide yields a modification containing reactivepyridyl disulfide groups in a single step. A pyridyl disulfide willreadily undergo an interchange reaction with a free sulfhydryl to yielda single mixed disulfide product.

In some cases, sulfhydryl groups activated with the leaving group5-thio-2-nitrobenzoic acid can be used to couple free thiols bydisulfide interchange similar to pyridyl disulfides, as describedherein. The disulfide of Ellman's reagent readily undergoes disulfideexchange with a free sulfhydryl to form a mixed disulfide withconcomitant release of one molecule of the chromogenic substance5-sulfido-2-nitroben-zoate, also called 5-thio-2-nitrobenzoic acid(TNB). The TNB-thiol group can again undergo interchange with asulfhydryl-containing target molecule to yield a disulfide crosslink.Upon coupling with a sulfhy-dryl compound, the TNB group is released.

In some cases, disulfide reduction may be performed usingthiol-containing compounds such as TCEP, DTT, 2-mercaptoethanol, or2-mercaptoethylamine.

In some cases, a vinyl sulfone group of a biomolecule or solid supportmay be utilized for bioconjugation. For example, the Michael addition ofthiols to activated vinyl sulfones to form biomolecule-materialconjugates have been used to demonstrate that cysteine capped peptidescould cross-link vinyl-sulfone functionalized multiarm PEGs to formprotease responsive hydrogels, enabling cell invasion during tissuegrowth. In some cases, in addition to thiols, vinyl sulfone groups canreact with amines and hydroxyls under higher pH conditions. The productof the reaction of a thiol with a vinyl sulfone gives a singlestereoisomer structure. In addition, crosslinkers and modificationreagents containing a vinyl sulfone can be used to activate surfaces ormolecules to contain thiol-reactive groups.

In some cases, thiol-containing biomolecules can interact with metalions and metal surfaces to form dative bonds for bioconjugation. In somecases, oxygen- and nitrogen-containing organic or biomolecules may beused to chelate metal ions, such as in various lanthanide chelates,bifunctional metal chelating compounds, and FeBABE. In addition, aminoacid side chains and prosthetic groups in proteins frequently formbioinorganic motifs by coordinating a metal ion as part of an activecenter

In some cases, thiol organic compounds may be used routinely to coatmetallic surfaces or particles to form biocompatible layers or createfunctional groups for further conjugation of biomolecules. For instance,thiol-containing aliphatic/PEG linkers have been used to formself-assembled monolayers (SAMs) on planar gold surfaces and particles.

In some cases, a number of alternative coupling systems may be used forbiomolecule functionalization. These include the use of O-nitrophenylesters (which possess reduced stability in aqueous conditions) or1,1′-carbonyldiimidazole (CDI) to form amine-bridging carbamate linkagesrather than amides. Hydrazines can also be used in place of aminesduring EDC/NHS mediated couplings. Hydrazine-functionalized peptides canbe coupled to biomaterials in a single step at pH 5-6. In doing so, adegree of site-selectivity can be achieved over lysine residues present.This approach has been successfully implemented by Madl and co-workersto conjugate reactive groups to alginate hydrogels, enabling indirectfunctionalization with growth factors and adhesion peptides.

In some cases, N-terminal modification of a biomolecule may be utilizedfor bioconjugation. For example, 2-pyridinecarboxaldehyde modifiedacrylamide hydrogels may react specifically with the N-terminus of ECMproteins, forming a cyclic imidazolidinone product with the adjacentamide bond and enabling the orientated display of these keybioinstructive motifs.

In some cases, acrylates, acrylamides, and methacrylates of abiomolecule or solid support may be utilized for bioconjugation. In somecases, thiol-ynes of a biomolecule or solid support may be utilized forbioconjugation.

In some cases, thiol-reactive conjugation such as native chemicalligation (NCL) can be utilized to attach peptides and proteins tobiomaterial scaffolds via peptide bond formation. For example, a peptidehaving a C-terminal thioester reacts with an N-terminal cysteine residuein another peptide to undergo a trans-thioesterification reaction, whichresults in the formation of an intermediate thioester with the cysteinethiol.

In some cases, strong binding of (strept)avidin may be used for thesmall molecule biotin for bioconjugation. In some cases, (strept)avidinand biotin may be attached to a biomolecule or solid support forbioconjugation. In some cases, modification reagents can add afunctional biotin group to proteins, nucleic acids, and otherbiomolecules. In some cases, depending on the functionality present onthe biotinylation compound, specific reactive groups on antibodies orother proteins may be modified to create a (strept)avidin binding site.Amines, carboxylates, sulfhydryls, and carbohydrate groups can bespecifically targeted for biotinylation through the appropriate choiceof biotin derivative. In some cases, photoreactive biotinylationreagents are used to add nonselectively a biotin group to moleculescontaining no convenient functional groups for modification. In somecases, biotin-binding proteins can be immobilized onto surfaces,chromatography supports, microparticles, and nanoparticles for use incoupling biotinylated molecules. In some cases, a series of(strept)avidin-biotin interactions can be built upon each other toutilize the multivalent nature of each tetrameric (strept)avidinmolecule and enhance the detection capability for the target. In somecases, amine-reactive biotinylation reagents that may contain reactivegroups off biotin's valeric acid side chain are able to form covalentbonds with primary amines in proteins and other molecules. In somecases, NHS esters spontaneously react with amines to form amide linkageswhereas carboxylate-containing biotin compounds can be coupled to aminesvia a carbodiimide-mediated reaction using EDC. In some cases,NHS-iminobiotin can be used to label amine-containing molecules with animinobiotin tag, providing reversible binding potential with avidin orstreptavidin. In some cases, Sulfo-NHS—SS-biotin (also known asNHS—SS-biotin) issulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate, along-chain cleavable bio-tinylation reagent that can be used to modifyamine-containing proteins and other molecules. In some cases,1-biotinamido-4-[4′-(maleimidomethyl) cyclohexane-carboxamido]butane, abiotinylation reagent containing a maleimide group at the end of anextended spacer arm reacts with sulfhydryl groups in proteins and othermolecules to form stable thioether linkages. In some cases,N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide where thereagent contains a 1,6-diaminohexane spacer group which is attached tobiotin's valeric acid side chain, the terminal amino group of the spacermay be further modified via an amide linkage with the acid precursor ofSPDP to create a terminal, sulfhydryl-reactive group. The pyridyldisulfide end of biotin-HPDP may react with free thiol groups inproteins and other molecules to form a disulfide bond with loss ofpyridine-2-thione.

In some cases, a carboxylate of a biomolecule or solid support may beutilized for bioconjugation. In some cases, diazomethane and otherdiazoalkyl derivatives may be used to label caroxylate groups. In somecases, N,N′-Carbonyl diimidazole (CDI) may be used to react withcarboxylic acids under nonaqueous conditions to form N-acylimidazoles ofhigh reactivity. An active carboxylate can then react with amines toform amide bonds or with hydroxyl groups to form ester linkages. Inaddition, activation of a styrene/4-vinylbenzoic acid copolymer with CDImay be used to immobilize an enzyme lysozyme or other biomoleculethrough its available amino groups to the carboxyl groups on to amatrix.

In some cases, carbodiimides function as zero-length crosslinking agentscapable of activating a carboxylate group for coupling with anamine-containing compound for bioconjugation or a solid support. In somecases, carbodiimides are used to mediate the formation of amide orphosphoramidate linkages between a carboxylate and an amine or aphosphate and an amine.

In some cases, N,N′-disuccinimidyl carbonate or N-hydroxysuccinimidylchloroformate may be utilized in bioconjugation. N,N′-Disuccinimidylcarbonate (DSC) consists of a carbonyl group containing, in essence, twoNHS esters. The compound is highly reactive toward nucleophiles. Inaqueous solutions, DSC will hydrolyze to form two molecules ofN-hydroxysuccinimide (NHS) with release of one molecule of CO₂. Innonaqueous environments, the reagent can be used to activate a hydroxylgroup to a succinimidyl carbonate derivative. DSC-activated hydroxyliccompounds can be used to conjugate with amine-containing molecules toform stable crosslinked products.

In some cases, sodium periodate can be used to oxidize hydroxyl groupson adjacent carbon atoms, forming reactive aldehyde residues suitablefor coupling with amine- or hydrazide-containing molecules forbioconjugation. For example, these reactions can be used to generatecrosslinking sites in carbohydrates or glyco-proteins for subsequentconjugation of amine-containing molecules by reductive amination.

In some cases, enzymes may be used to oxidize hydroxyl-containingcarbohydrates to create aldehyde groups for bioconjugation. For example,the reaction of galactose oxidase on terminal galactose orN-acetyl-d-galactose residues proceeds to form C-6 aldehyde groups onpolysaccharide chains. These groups can then be used for conjugationreactions with amine- or hydrazide-containing molecules.

In some cases, reactive alkyl halogen compounds can be used tospecifically modify hydroxyl groups in carbohydrates, polymers, andother biomolecules for bioconjugation.

In some cases, an aldehyde or ketone of a biomolecule or solid supportmay be used for bioconjugation. For example, derivatives of hydrazine,especially the hydrazide compounds formed from carboxylate groups, canreact specifically with aldehyde or ketone functional groups in targetbiomolecules. To further stabilize the bond between a hydrazide and analdehyde, the hydrazone may be reacted with sodium cyanoborohydride toreduce the double bond and form a secure covalent linkage.

In some cases, an aminooxy group of a biomolecule or solid support maybe used for bioconjugation. For example, the chemoselective ligationreaction that occurs between an aldehyde group and an aminooxy groupyields an oxime linkage (aldoxime) that has been used in manybioconjugation reactions, as well as in the coupling of ligands toinsoluble supports including surfaces. This reaction is also quiteefficient with ketones to form an oxime called a ketoxime.

In some cases, cycloaddition reactions may be utilized forbioconjugation. In cycloaddition reactions for bioconjugation, two ormore unsaturated molecules are brought together to form a cyclic productwith a reduction in the degree of unsaturation, these reaction partnersrequired are typically absent from natural systems, and so the use ofcycloadditions for conjugation requires the introduction of unnaturalfunctionality within the biomolecule coupling partner.

In some cases, Copper-Catalyzed Azide-Alkyne Cycloadditions may beutilized for bioconjugation. In some cases, the (3+2) cycloadditionbetween an azide and alkyne proceeds spontaneously at high temperatures(>90° C.), producing a mixture of two triazole isomers. In some cases,this reaction proceeds at room temperature, ambient, oxygenated, and/oraqueous environments. In some cases, for example, the formation ofpeptide-material conjugates by CuAAC, using alkyne-capped peptides toform hydrogels with azide-functionalized PEG. In some cases, CuAAC hasbeen widely used to functionalize scaffolds with alkyne and azidefunctionalized peptides and carbohydrates, in part due to the ease withwhich the amino acids azidolysine and homopropargylglycine can beintroduced by solid-phase peptide synthesis. In some cases, To achievebiomaterial conjugation via CuAAC, the required copper(I) catalyst caneither be added directly, or generated in situ by reduction of aninitial copper(II) complex, most commonly using ascorbic acid. Theaddition of a reducing agent further reduces the sensitivity of theCuAAC ligation to oxygen. Although no additional ligand is necessary fortriazole formation, the addition of tertiary amine based ligands may beused.

In some cases, Strain-Promoted Azide-Alkyne Cycloadditions (SPAAC) maybe utilized for bioconjugation. In some cases, highly strainedcyclooctynes react readily with azides to form triazoles underphysiological conditions, without the need for any added catalyst. Insome cases, in addition to the use of SPAAC for peptide conjugation, anumber of prominent reports have used SPAAC to conjugate proteinsubstrates to cyclooctyne functionalized biomaterials via theintroduction of an unnatural azide motif into the protein couplingpartner. In some cases, for example, this is achieved by includingmaleimide functionalization of native cysteines present in bonemorphogenetic protein-2 (BMP-2), via enzyme-mediated N-terminalmodification of IFN-7, or via codon reassignment with the unnaturalamino acid 4-azidophenylalanine in a number of protein substrates. Insome cases, supramolecular host-guest interactions can also be used topromote azide-alkyne cycloaddition. For example, by bringing tworeactive partners into close proximity within the cavity of acucurbit[6]uril host, efficient cycloaddition could be achieved on thesurface of proteins, this strategy may be extended to other appropriatebiomolecules.

In some cases, inverse-electron demand Diels-Alder reactions (IEDDA) maybe utilized for bioconjugation. For example, the inverse-electron demandDiels-Alder (IEDDA) reaction between 1,2,4,5-tetrazines and strainedalkenes or alkynes may be employed. A wide range of suitable derivativesfor undertaking biomolecule conjugation have been reported, for example,a series of increasingly strained (and thus reactive) trans-cyclooctenesmay be utilized. In some cases, functionalized norbornene derivativesmay be utilized for undertaking IEDDA reactions. In some cases,triazines may be utilized. In some cases, spirohexene may be utilized.These strategies may be extended to other appropriate biomolecules. Insome cases, hetero-Diels-Alder cycloaddition of maleimides and furansmay be utilized for bioconjugation. For example, the coupling offuran-functionalized RGDS peptides to maleimide-functionalizedPEG-hydrogels may be utilized, this strategy may be extended to otherappropriate biomolecules. In some cases, furan-functionalizedhyraluronic acid hydrogels can be cross-linked with adimaleimide-functionalized peptide via Diels-Alder cycloaddition.MMP-cleavable peptides enable the migration of seeded cancer through thegel.

In some cases, oxime and hydrazone formation may be utilized forbioconjugation. In some cases, the stable attachment of peptides and DNAto biomaterials via hydrazone formation can be achieved via difunctionalcross-linking, this strategy may be extended to other appropriatebiomolecules. In some cases, the attachment of ketone or aldehydemodified green fluorescent protein (GFP) or metallothionein tohydroxylamine-functionalized synthetic polymers may be extended to otherappropriate biomolecules. For example, protein cross-linked hydrogelswere produced through oxime modification at both the protein N- andC-termini.

In some cases, the Diels-Alder reaction consists of the covalentcoupling of a diene with an alkene to form a six-membered ring complexfor bioconjugation.

In some cases, transition metal complexes may be utilized forbioconjugation. The nature of late transition metals may make atransition metal complex well suited to the manipulation of unsaturatedand polarizable functional groups (olefins, alkynes, aryl iodides,arylboronic acids, etc.). For example, Pd(O)-functionalized microspheresmay mediate allyl carbamate deprotections and Suzuki-Miyauracross-coupling in the cytoplasm. In other examples, a ruthenium catalystmay be used to mediate allyl carbamate deprotection of a cagedfluorophore inside living cells. In some cases, applications ofpalladium-based applications in cell culture include copper-freeSonagashira coupling, extracellular Suzuki coupling on the surface of E.coli cells, and conjugation of thiol groups with allyl selenosulfatesalts. In some cases, olefin metathesis may be utilized forbioconjugation. For example, with ruthenium complexes, S-allylcysteinecan be easily introduced into proteins by a variety of methods,including conjugate addition of allyl thiol to dehydroalanine, directallylation of cysteine, desulfurization of allyl disulfide, or metabolicincorporation as a methionine surrogate in methionine auxotrophic E.coli.

In some cases, complex formation with boronic acid derivatives may beused for bioconjugation. For example, boronic acid derivatives are ableto form ring structures with other molecules having neighboringfunctional groups consisting of 1,2- or 1,3-diols, 1,2- or 1,3-hydroxyacids, 1,2- or 1,3-hydroxylamines, 1-2- or 1,3-hydroxyamides, 1,2- or1,3-hydroxyoximes, as well as various sugars or biomolecules containingthese species.

In some cases, enzyme-mediated conjugation may be utilized forbioconjugation. For example, the transglutaminase enzyme familycatalyzes the formation of isopeptide bonds between the primary amine oflysine side chains and the amide bonds of a complementary glutamineresidue, this strategy may be extended to other appropriatebiomolecules. In other cases, peroxidase-mediated conjugation may beutilized for bioconjugation. For example, horse radish peroxidase (HRP)may be utilized to oxidize a wide range of organic substrates such asphenol group of tyrosie to generate a highly reactive radical or quinoneintermediate that undergoes spontaneous dimerization, resulting in theformation of an ortho carbon-carbon bond between two tyrosine residues,this strategy may be extended to other appropriate biomolecules. In somecases short peptide tags may be utilized for bioconjugation. Thesepeptide tags may be as short as 5 amino acids long and may be appendedto a peptide or protein substrate which allows for their subsequentmodification.

In some cases, polymerization of low molecular weight monomers may beutilized for bioconjugation. Polymerization may be classified asproceeding via one of two mechanisms, either chain-growth orstep-growth. During chain-growth polymerization, monomers are added atthe “active” end of a growing polymer chain, resulting in the formationof high molecular weight materials even at low conversions. Duringstep-growth polymerizations short oligomer chains couple to formpolymeric species, requiring high conversions in order to reach highmolecular weights. Both techniques can be used to formbiomolecule-polymer conjugates. The polymerization of acrylate andmethacrylate monomers has proven particularly fruitful. For example,acrylate and methacrylate modified peptides and glycans can be readilypolymerized. Similarly, availability of the synthetic oligonucleotidephosphoramidite building block “Acrydite”, free-radical polymerizationremains one of the most common methods through which to form DNA and RNAfunctionalized biomaterials. By undertaking polymerization in thepresence of a comonomer, the density of biomolecule presentation can beeasily tuned, allowing potential difficulties from steric hindrance tobe overcome. Initiation of polymerization can be triggered by a numberof means, including heat, UV and visible light, redox reactions, andelectrochemistry. Acrylate modified proteins can also undergopolymerization to produce functional materials, while retainingbiological activity. In some cases living radical polymerizations (LRPs)may be utilized for bioconjugation. For example, the most commonly usedLRPs for the formation of bioconjugates include atom-transfer radicalpolymerization (ATRP), reversible addition-fragmentation chain transfer(RAFT) polymerization, and nitroxide-mediated polymerization (NMP).

In some cases, photoconjugation may be utilized for bioconjugation. Insome cases polymerization is initiated by the production of a radicalspecies, which then propagates through bond formation to create anactive polymer chain. The initiation step can be induced via a number ofstimuli, with thermal decomposition, redox activation, andelectrochemical ionization of an initiating species being among the mostcommon. Alternatively, many initiators can be activated vialight-induced photolytic bond breakage (type I) or photoactivatedabstraction of protons from a co-initiator (type II). Photoinitiationoffers the benefits of being applicable across a wide temperature range,using narrow and tunable activation wavelengths dependent on theinitiator used, rapidly generating radicals, and the ability to controlpolymerization by removing the light source. Importantly, the toleranceof polymerizations to oxygen is greatly enhanced, enablingpolymerization in the presence of cells and tissues. The incorporationof acrylate-functionalized peptides and proteins duringphotopolymerization may be used as a method for producing biomaterialconjugates. Alternatively, the photoinitiated attachment of polypeptidesto pendant vinyl groups on preformed materials has also been widelyreported and more recently used for 3D patterning via two-photonexcitation. A wide range of photoinitators may be used inphotoconjugation conjugations. For example but not limited to, Eosin Y,2,2-dimethoxy-2-phenyl-acetophenone, Igracure D2959, lithiumphenyl-2,4,6-trimethylbenzoylphosphinate, and riboflavin may be used asphotoinitiators. Photoinitiators generally absorb light to initiate thephotoreaction processes. In some cases, photoconjugation may utilize aphoto thiol-ene reaction. Thiols can also react with alkenes via afree-radical mechanism. A thiol radical first reacts with an alkene togenerate a carbon-centered radical, which can then abstract a protonfrom another thiol and thus propagate the reaction. Photo thiol-enereactions may be accelerated by electron-rich alkenes, which generateunstable carbon-radical intermediates able to rapidly abstractthiol-hydrogens. Exceptions to this rule are norbornene derivatives, inwhich reactivity is driven instead by the release of ring strain uponthiol addition. This leads to a general trend in reactivity ofnorbornene>vinyl ether>propenyl>allyl ether>acrylate>maleimide.Norbornenes and allyloxycarbonyls (alloc groups) have been particularlywidely used for peptide/protein-biomaterial functionalization, due tothe almost negligible contribution of chain transfer and their ease ofintroduction during peptide synthesis, respectively. For example, analloc group, typically used as an orthogonal lysine protecting groupduring solid-phase peptide synthesis, is an efficient photo thiol-enereactive handle. In other examples, norbornene photo thiol-ene reactionsmay be used for the tethering and spatial patterning of bioactivepeptides and growth factor proteins. In addition to the most commonlyused alloc and norbornene reactive groups, other alkenes have also beenused for biomaterial functionalization. For example, codon reassignmenthas been used to site-specifically incorporate allyl-cysteine residuesinto proteins, which can subsequently undergo conjugation through theuse of photo thiol-ene reactions. Alternatively, acrylates can undergomixed-mode photopolymerizations in the presence of cysteine cappedpeptides, while allyl disulfide structures have recently been shown toundergo reversible and controlled exchange of conjugated thiols.

In some cases, aryl azide or halogenate aryl azides of a biomolecule orsolid support may be utilized for bioconjugation.

In some cases, photoreactive group benzophenone may be utilized forbioconjugation.

In some cases, photoreactive group anthraquinone may be utilized forbioconjugation.

In some cases, photo thiol-yne reactions may be utilized forbioconjugation. Most examples of photo thiol-yne reactions haveexploited simple propargyl-ether or -amine reactive handles.

In some cases, photocaging and activation of reactive functionalitiesmay be utilized for bioconjugation. Generally, a transient reactivespecies is formed whether it be an acrylate or thiol derived radical. Insome cases, photocaging may be used to mask or protect a functionalgroup until it is desirable for it to be exposed. In some cases, themost widely utilized cages are based around o-nitrobenzyl and coumarinchromophores. For example, nitrobenzyl-capped cysteine residues may bedecaged by irradiation with 325 nm UV light, the released thiol may thenreact with maleimide-functionalized peptides via Michael addition, togenerate a patterned hydrogel able to guide cell migration. In somecases, 6-bromo-hydroxycoumarins may be used for thiol-caging. In somecases, photoaffinitiy probes may be utilized for bioconjugation where ahighly reactive intermediate upon irradiation, which then reacts rapidlywith the nearest accessible functional group with high spatialprecision. In some cases, the most commonly used are phenylazides,benzophenones, and phenyl-diazirines. In some cases, photocagedcycloadditions may be used. For example, the UV irradiation oftetrazoles has been shown to generate a reactive nitrile-imineintermediate which can undergo rapid cycloaddition withelectron-deficient alkenes such as acrylates or acrylamides. In somecases, the nitrile-imine side-reactivity with thiols may be utilized forsite-specifically conjugate cysteine containing proteins to tetrazolefunctionalized surfaces.

In some cases, noncovalent interactions may be utilized forbioconjugation. In some cases, noncovalent binding plays a vital role incells, controlling biomolecular interfaces and influencingprotein-protein interactions, DNA-DNA complexation, DNA-proteininterfaces, protein localization, and more. In some cases, noncovalentsequences which display a binding affinity for the biomolecule ofinterest, allow for postfabrication modification or for nativebiomolecules to be simply sequestered from the surroundings withinbiological samples. The most commonly used binding sequences are shortpeptides between 7 and 20 amino acids in length, derived from a varietyof sources, including known protein binding domains present in vivo ordetermined through techniques such as phage display. In some cases,short oligonucleotides known as aptamers can also be used to bind avariety of protein substrates, including the cytokines vascularendothelial growth factor (VEGF) and platelet derived growth factor(PDGF), as well as cell surface proteins such as epidermal growth factorreceptor (EGFR). In some cases, binding sequences can also be introducedinto a biomaterial with affinity for native biopolymers, such asheparin. In some cases, by first inducing biopolymer binding, theadsorption of an added or endogenous growth factor or signaling proteinto a biomaterial scaffold can then be controlled. In some cases, bindingaffinity at the amino acid level can also be exploited to enable peptideand protein conjugation to certain biomaterial substrates. For example,the binding of unnatural catechol-based amino acids can be used toinduce binding to metal oxide containing bioglasses and metallicimplants, enabling the bioactivity of these important technologies to beenhanced.

In some cases, self-assembling peptides may be utilized forbioconjugation. For example, native peptides and proteins adopt a seriesof secondary structures, including β-sheets and α-helices, which canboth stabilize individual sequences and control interproteinaggregation. In some cases, self-assembling peptides have been usedextensively to assemble hydrogels and fibrous materials. In many ofthese structures, biological epitopes or functional groups can beappended to some or all of the peptide building blocks during peptidesynthesis, to add the desired bioactivity into the system.Peptide-ligands ranging from simple adhesion motifs, to laminin derivedepitopes, and growth factor mimetics have all been displayed on thesurface of self-assembled fibrils. Alternatively, glycopeptides can beassembled in order to recruit extracellular signaling proteins andgrowth factors, mimic glycosylation patterns within hyaluronic acid, orinvestigate optimal sulfonation ratios in glycosaminoglycan scaffolds.In some cases, self-assembling domains can also be added to full-lengthproteins, leading to the incorporation of pendant functionality duringhydrogel formation. In some cases, the propensity of peptides to formsecondary structures has also been exploited within nonself-assemblingscaffolds. This may be achieved by mixing a self-assembling peptide intoa covalent hydrogel, composed of either a noninteracting polymer such asinterpenetrating networks of PEG or systems where additional chargeinteractions further stabilize the final construct, for example betweenpositively charged peptides and negatively charged alginate gels. As analternative, pendant helical groups can be attached to a covalentmaterial and used to drive the noncovalent attachment of bioactivegroups such as growth factors via self-assembly into coiled-coil triplehelices.

In some cases, host-guest chemistry may be utilized for bioconjugation.For example, the adhesive properties of a β-cyclodextrin modifiedalginate scaffold could be controlled in situ through the addition of aguest naphthyl-functionalized RGDS peptide and by subsequentlyintroducing a non-cell adhesive adamantane-RGES peptide with a higherhost binding constant, dynamic modulation of fibroblast cell attachmentwas enabled. Host-guest interactions between cyclodextrin and naphthyl-or adamantane-functionalized peptides allow alginate functionalization,this may be applied to other appropriate biomolecules.

In some cases, biotin-(strept)avidin may be utilized for bioconjugation.For example, avidin and streptavidin are homotetrameric proteins thatcan simultaneously bind up to four molecules of their small moleculebinding partner biotin. The small size of biotin (with a mass of just244 Da) and the ease with which it can be functionalized via its freecarboxylic acid has led to biotin-(strept)avidin binding findingwidespread use as a means to undertake biomaterial conjugation.Streptavidin-protein fusions can be produced recombinantly and bound tosuitably functionalized surfaces to achieve conjugation. In some cases,biomolecule biotinylation is undertaken, and this construct is thenbound to a (strept)avidin functionalized surface. In some cases, thiscan either be achieved by a direct route, via chemical preconjugation ofthe material with (strept)-avidin, or by exploiting the tetramericbinding of (strept)avidin to mediate indirect modification orcross-linking of biotin-functionalized scaffolds.

In some cases, nucleic acids may be utilized for bioconjugation. In somecases, in an analogous fashion to self-assembling peptides, nucleicacids can also form assembled materials themselves, to generate tunableplatforms for the display of biomolecules. In some cases, DNA-taggedpeptides and growth factors can be conjugated to a suitablyfunctionalized biomaterial and used to elicit a desired biologicaleffect on a localized cell population.

Generally, incorporating reactive handles may be utilized forbioconjugation. For example, introducing uniquely reactive motifs intobiomolecule substrates provides a chemical “tag” which allowssingle-site selectivity or specificity to be achieved. In some cases,short peptides and oligonucleotides can typically be produced via solidphase synthesis (SPS). The versatility of organic synthesis allowsdifficulties in reactive handle incorporation to be overcome, with awide range of suitably functionalized amino acids and oligonucleotidesavailable as described herein. In some cases, an alternative approach isto introduce unnatural amino acids (UAAs) bearing the desired reactivehandles. This may be achieved via the modification of lysine residueswith amine-reactive derivatives. In some cases, the use of auxotrophicbacterial strains, which are unable to biosynthesise a particular aminoacid and thus require uptake from the growth media, by starving thebacteria of the native amino acid and supplementing it with astructurally related unnatural analogue, the bacterial cells can willincorporate the UAA during translation. This technique may be used toinstall azide- and alkyne-based mimics of methionine, leading to theintroduction of reactive handles for undertaking CuAAC and SPAACreactions. Analogous strategies can be used for the incorporation ofunnatural monosaccharides, enabling the remodelling of complex glycans.In some cases, the use of codon reassignment using orthogonal tRNA andtRNA synthetase pairs that selectively recognize and charge an UAAduring translation. In some cases, this may be achieved by reassigningthe amber stop-codon, UAG, by incorporating a tRNA_(CUA)/tRNA synthetasepair from an alternative kingdom into the host cell. This pair may beable to install the desired UAA, while being effectively invisible tothe endogenous cell machinery. As a result, site-directed mutagenesiscan be used to introduce a single TAG codon at the desired position ofthe coding DNA, leading to the singular introduction of the UAA withhigh specificity and selectivity.

In some cases, one or more functional groups may release a reporter whenreacted with another functional group, or with a SNAP or biologicalentity or chemical entity. Having a reporter released when the SNAP andbiological or chemical entity are conjugated may allow tracking of thereaction. In some cases, it may be possible to monitor the degree ofcompletion of a SNAP-biological/chemical entity conjugation reaction bymonitoring the concentration of free reporter. In some cases, thereporter may fluoresce once released by the conjugation reaction.

In some cases, the biological or chemical entity may be functionalizedwith a linker. In some cases, functionalizing the biological or chemicalentity with a linker may decrease steric hindrance. A linker maycomprise a rigid or semi-rigid moiety which can hold the biological orchemical entity away from the SNAP. In some cases, the linker may be along, moderate or short linker. In some cases, the linker may compriseone or more component selected from PEG, DNA, short carboxyl, carbonchain, peptoid, spacer, and/or glycer, among other examples.

In some cases, the SNAPs, seeds, and/or biological or chemical entitiesmay be functionalized using single pot proteomics methods. Single potproteomics methods may result in very high efficiency offunctionalization. In some cases, single pot proteomics methods may beuseful to functionalize biological or chemical entities with very lowlevels of loss of the entities.

In some embodiments, a SNAP is a polymer which may be grown from theseed. For example if the seed is a DNA oligonucleotide then the SNAP maybe a DNA molecule. In some cases, the SNAP may be a DNA molecule withregions of internal complementarity such that the molecule mayself-hybridize. For example, the SNAP may be a DNA cluster, formed byself hybridization within the molecule. In some cases, the SNAP may beformed from DNA, RNA, L-DNA, L-RNA, LNA, PNA, or a mixture of two ormore different types of nucleic acid. In some cases, the SNAP may have arepeating structure, such as a repeating sequence of nucleotides. Insome cases, the SNAP may be an irregular polymer without a repeatingsequence. For example, the SNAP may comprise a random sequence ofnucleotides.

In some cases, a SNAP may be formed by rolling circle amplification. Aplasmid, or other circular nucleic acid molecule, may be provided as atemplate, together with a primer that binds to the circular nucleic acidmolecule, wherein the primer comprises a functional group on the 5′ end.Performing a polymerase chain reaction (PCR) with a sufficiently longextension step, or merely a polymerase extension reaction, will allowthe functionalized primer to bind the circular nucleic acid molecule andproduce a single stranded nucleic acid product. The length of the singlestranded nucleic acid product may be influenced by altering theextension time, the polymerase enzyme used, or the reaction conditions.In some cases, the circular nucleic acid template contains regions ofinternal complementarity, such that the single stranded nucleic acidproduct will contain regions which may self-hybridize. In some cases,the circular nucleic acid template is a dsDNA molecule. In some cases,the single stranded nucleic acid product is an ssDNA molecule. In somecases, the polymerase used is a DNA polymerase.

In some cases, a SNAP may be formed by nucleic acid origami, or DNAorigami. DNA origami generally refers to the nanoscale folding of DNA tocreate non-arbitrary two- and three-dimensional shapes at the nanoscale.The specificity of the interactions between complementary base pairs canmake DNA a useful construction material. In some cases, the interactionsbetween different regions may be controlled through design of the basesequences. DNA origami may be used to create scaffolds that hold othermolecules in place or to create structures all on its own.

SNAPs as described herein can include those created via nucleic acidorigami. Commonly, nucleic acid origami can refer to DNA origami, but itcan also refer to RNA origami, origami of a combination of DNA and RNAmolecules, or origami of nucleic acid molecules which can be other thanDNA or RNA, such as a silicon-based nucleic acid, among other examples.Nucleic acid origami can result in a nucleic acid molecule which has anengineered shape. The engineered shape can be a shape which has beenpartially or fully planned. The planning of the shape can compriseplanning or engineering what sections of nucleic acid bind, where asegment of nucleic acid can fold, where a segment of nucleic acid can besingle stranded, where a segment of nucleic acid can be double stranded,where a segment of nucleic acid can be bound to a segment of nucleicacid of the same strand, or where a segment of nucleic acid can be boundto a segment of nucleic acid on another strand. In some cases,non-nucleic acid molecules, such as protein, can be used to encouragenucleic acid into the engineered shape.

Generally, nucleic acid origami can comprise at least one or more longnucleic acid strand and one or more short nucleic acid strands.Commonly, these nucleic acid strands are single stranded, although theycan have segments which can be double stranded. One of the short strandscan comprise at least a first segment which can be complementary to afirst segment of the long strand, as well as a second segment which canbe complementary to a second segment of the long strand. When the shortand long strands are incubated under conditions that can allowhybridization of nucleotides, the shorter oligonucleotide can hybridizewith the longer oligonucleotide. This hybridization can give shape tothe nucleic acid molecule. For example, if the two segments on the firststrand are separated, then these two segments can be brought togetherduring hybridization to create a shape. In some cases, a short strandcan bind to at least 2, 3, 4, 5, or 6 segments which can bind to atleast 2, 3, 4, 5, or 6 complementary segments of the long nucleic acidstrand.

In some cases, a short strand can have one or more segments which can benot complementary to the long strand. In such a case, the segment whichis not complementary to the long strand can be at least about 1, 2, 3,4, 5, 10, 15, or 20 nucleotides long.

This process can be performed with at least about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,or more short nucleic acid strands. These short nucleic acid strands caneach bind to one or more different segments of the long nucleic acidstrand. Each short nucleic acid strand which hybridizes to the longnucleic acid strand can lead to a fold in the long nucleic acid strand.In some cases, the number of short strands can be correlated with thecomplexity of the engineered shape. For example, an engineered shapewith many folds can utilize more short nucleic acid strands than anengineered shape with few folds. An engineered shape can have at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250, 300, or more folds.

In some cases, more than one long strand can be incorporated into thenucleic acid origami structure. This can be done for example to increasethe complexity of the engineered shape, to ease the designing orplanning of the engineered shape, to avoid the creating of a shape whichis more thermodynamically stable than the desired engineered shape, tomake the creation of the engineered shape easier, or to manage costs ofcreating the engineered shape.

Incorporation of more than one long strand can be accomplished bydesigning the 2 or more long strands such that each strand has at leastone segment that can be complimentary to a segment of the other strand,or by designing the 2 or more long strands such that each has at leastone segment which can be complementary to a region of a short nucleicacid strand, such that both long strands have segments complementary tothe short nucleic acid strand.

Short nucleic acid strands can have complementarity to one long nucleicacid strand or more than one long nucleic acid strand. In some cases, ashort nucleic acid strand can also have complementarity to one or moreshort nucleic acid strands.

The terms “long” and “short” herein are meant to be general terms. Along strand can be longer than a short strand, although in someinstances a long strand can be the same size as a short strand. In somecases, a long strand can be at least about 30, 40, 50, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides long.In some cases, a short strand can be at least about 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more nucleotideslong.

An engineered shape can be designed for a specific purpose. For example,an engineered shape can be designed to support a load, encapsulate amolecule, bind a molecule, connect two or more molecules, fit into acavity, bind a protuberance, or other purpose. An engineered shape canany shape, such as oblong, rectangular, round, circular, spherical,flat, textured, smooth, symmetrical, asymmetrical, conical, orirregular. An engineered shape can be a cube, pyramid, box, cage,ladder, or tree.

An engineered shape or SNAP formed via nucleic acid origami as describedabove can be assembled. Assembly can refer to the process by which thenucleic acid strands hybridize to each other to create the engineeredshape.

An engineered shape or SNAP can be spontaneously self-assembling.Self-assembly can occur when long and short oligonucleotides havingregions which can be complimentary are incubated together. Duringspontaneous self-assembly, the nucleotides can hybridize and theengineered shape can be created during incubation without the help of ahelper molecule or catalyst. Such self-assembling can occur underspecific conditions or a range of specific conditions. Conditions whichcan be considered when incubating DNA strands for self-assembly can besalt concentration, temperature, and time.

Sometimes, assembly can utilize or require a catalyst. In such cases,the catalyst can speed up assembly or ensure the assembly results in aparticular desired engineered shape. A catalyst can comprise RNA, DNA,or protein components.

The salt concentration during assembly can be less than 1 M, less than0.5M, less than 0.25 M, less than 0.1M, less than 0.05 M, less than 0.01M, less than 0.005 M, or less than 0.001 M.

The temperature during assembly can be at least room temperature. Insome cases, the temperature during assembly can be at least about 50,60, 70, 80, 85, 90, or 95° C. In some cases, the temperature duringassembly can vary. For instance, the temperature can be increased to atleast about 20, 30, 40, 50, 60, 70, 80, 85, 90, or 95° C. This increasecan ensure the nucleic acid strands do not comprise a secondarystructure prior to assembly. Once the temperature is increased asdescribed, it can be decreased, for example to about 20, 30, 40, 50, 60,70, or 80° C. This decrease in temperature can allow the nucleic acidsto hybridize. In some cases, the decrease in temperature can occur overabout 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 45, or 60 minutes.

Assembly can be performed stepwise. In such cases, a subset of thenucleic acid molecules can be incubated together first. After thesemolecules are allowed to hybridize, one or more additional nucleic acidmolecules can be added and allowed to hybridize. In some cases, two ormore engineered shapes which have been assembled can be incubatedtogether for assembly into a larger engineered shape.

In some cases, assembly can comprise fractal assembly. Fractal assemblycan create a SNAP which can be an array of engineered shapes. Assemblycan occur in stages, which can simplify the design process or ensurecorrect assembly. Such an array can be assembled in at least 1, 2, 3, 4,5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,1000, or more stages. In some cases, the number of stages used cancorrelate with a reduction of spurious interactions. This can be due toa reduction in the total number of possible reactions at any given time.

SNAPs can be assembled into an array which can be at least 3×3, at least5×5, at least 10×10, at least 50×50, at least 100×100, or at least1000×1000 (engineered shapes×engineered shapes).

Each hybridization reaction can take about 10, 20, 30, 40, 50, or 60seconds. In some cases, each hybridization reaction can take about 1, 2,3, 4, 5, 10, 15, 20, 30, 40, 50, or 60 minutes. In some cases, ahybridization reaction can take more than 1 hour.

Nucleic acid origami may be used to preferentially choose how the SNAPwill “land” on the solid support. For example, nucleic acid origami maybe used to construct a SNAP with a landing surface that canpreferentially contact the solid support, A SNAP such as one made vianucleic acid origami can be designed to comprise a region that cancreate steric or electrostatic interactions with the support that caninfluence the orientation of the SNAP on the support. For example, theregion can comprise nucleotides having modifications e.g. to thebackbone of the nucleic acid which can promote interaction between theSNAP and the solid support. In further examples, the region can compriseprotuberances or cavities which can “fit” to cavities or protuberanceson the solid support. In some cases, the support surface can comprisechemical structuring (e.g. nanoparticles or oligonucleotides), clickreagents, or other rationally designed materials that can influence theposition and orientation of SNAP structures, including SNAPs synthesizedvia nucleic acid origami.

Nucleic acid origami can be used to construct a SNAP with a linker whichcan attach a biological or chemical entity, wherein the linker ispositioned relative to the landing surface such that the biological orchemical entity can be distal or approximately distal to the solidsupport. The linker may also comprise a region of dsDNA to force a rigidoutpost from the SNAP. In some cases, protein origami may also be used.

A surface can have properties such that a SNAP can bind to the surfacein such a way that it can flop or lean. The SNAP can flop or lean to theleft, to the right, to the front, to the back, or to any combination ofsides thereof. The SNAP can flop or lean once and remain in place, or itcan flop freely between sides over time. In some cases, the SNAP canpreferentially flop in one direction over one or more other directions.In some cases, the SNAP can preferentially avoid flopping in aparticular direction.

In some cases, for example, filamentous or stranded molecules, such asnanoparticles or oligonucleotide strands, can be attached to a surface.A SNAP, which can comprise an engineered shape, can comprise one or moremoieties which can bind to a filamentous or stranded molecule, such as adangling single stranded oligonucleotide or nanoparticle. Uponcontacting the surface with such SNAPs, the one or more moieties caninteract with one or more of the filamentous or stranded molecules. Insome cases, the moieties can bind tightly to the filamentous or strandedmolecules. The SNAPs can be removable or non-removable in such cases.

Computational modeling or simulation tools may be employed to design andoptimize oligonucleotide or protein sequences to create particular SNAPstructures.

In some cases, a SNAP may be a nucleic acid plasmid, such as a DNAplasmid. Plasmids may exist in a compact form known as supercoiled DNA.The radii of a supercoiled plasmid may be determined by the plasmidsize—i.e. a plasmid with a longer backbone will form a largersupercoiled entity. In some cases, a SNAP may comprise a plasmid with abackbone of between 5 kb and 150 kb. In some cases, a SNAP may comprisea plasmid with a backbone of between 5 kb and 100 kb. In some cases, aSNAP may comprise a plasmid with a backbone of between 5 kb and 90 kb.In some cases, a SNAP may comprise a plasmid with a backbone of between25 kb and 50 kb. In some cases, a SNAP may comprise a plasmid with abackbone of at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85kb, 90 kb, 95 kb, 100 kb, 105 kb, 110 kb, 115 kb, 120 kb, 125 kb, 130kb, 135 kb, 140 kb, 145 kb, or 150 kb. In some embodiments, SNAPs may beimaged using an imaging platform, such as Nanocyte or Leica.

In some cases, a SNAP may have a branched structure. For example theSNAP may be a dendrimer. Some examples of dendrimers may be found inNewkome, George R., and Carol D. Shreiner. “Poly (amidoamine),polypropylenimine, and related dendrimers and dendrons possessingdifferent 1->2 branching motifs: an overview of the divergentprocedures.” Polymer 49.1 (2008): 1-173. A dendrimer used with themethods of this disclosure may be a G1, G2, G3, G4, G5, G6, G7, G8, G9,G10, G11, G12, G13, G14, or G15 dendrimer. In some cases, the dendrimermay be higher than a G15 dendrimer, for example dendrimer between G15and G30.

In some embodiments, the SNAP may be a protein, or comprised ofproteins. For example the SNAP may be a protein fibril. The SNAP may becomprised of proteins known to form into fibrils, such as, for example,the tau protein, or portions of the tau protein. A 31 residue portion oftau which assembles into fibrils is described in Stohr, Jan, et al. “A31-residue peptide induces aggregation of tau's microtubule-bindingregion in cells.” Nature chemistry 9.9 (2017): 874. In some cases, theSNAP may comprise tetratricopeptide repeats. Examples oftetratricopeptide repeats may be found in Blatch, Gregory L., andMichael Lässle. “The tetratricopeptide repeat: a structural motifmediating protein-protein interactions.” Bioessays 21.11 (1999):932-939. Other examples of proteins which may assemble may be found inSpeltz, Elizabeth B., Aparna Nathan, and Lynne Regan. “Design ofprotein-peptide interaction modules for assembling supramolecularstructures in vivo and in vitro.” ACS chemical biology 10.9 (2015):2108-2115.

In some embodiments, the SNAP may be a single molecule. In someembodiments the SNAP may not be a single molecule. In some cases, theSNAP may be assembled from several molecules which bind non-covalently.For example the SNAP may be formed from two or more nucleic acidmolecules which hybridize together. In another example the SNAP may beformed from two or more protein molecules which assemble together vianon-covalent bonds.

In some embodiments, the SNAPs are between about 50 nm and about 100 umin diameter.

The SNAPs are generally polymeric molecules. These may be grown througha controlled polymerization reaction, a stepwise polymerizationreaction, or a step by step synthesis method. The growth of the SNAPsmay be controlled by the amount of monomers available, the length oftime the reaction is allowed to proceed, or the number of synthesissteps performed.

Each SNAP may have a diameter of at least about 10 nanometers (nm), orabout 10 nm, about 50 nm, about 75 nm, about 100 nm, about 125 nm, about150 nm, about 175 nm, about 200 nm, about 225 nm, about 250 nm, about275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about900 nm, about 925 nm, about 950 nm, about 975 nm, about 1000 nm, about1025 nm, about 1050 nm, about 1075 nm, about 1100 nm, about 1125 nm,about 1150 nm, about 1175 nm, about 1200 nm, about 1225 nm, about 1250nm, about 1275 nm, about 1300 nm, about 1325 nm, about 1350 nm, about1375 nm, about 1400 nm, about 1425 nm, about 1450 nm, about 1475 nm,about 1500 nm, about 1525 nm, about 1550 nm, about 1575 nm, about 1600nm, about 1625 nm, about 1650 nm, about 1675 nm, about 1700 nm, about1725 nm, about 1750 nm, about 1775 nm, about 1800 nm, about 1825 nm,about 1850 nm, about 1875 nm, about 1900 nm, about 1925 nm, about 1950nm, about 1975 nm, about 2000 nm, about 3000 nm, about 4000 nm, about5000 nm, about 6000 nm, about 7000 nm, about 8000 nm, about 9000 nm,about 10 m, about 15 m, about 20 m, about 25 m, about 30 m, about 40 m,about 50 m, about 75 m, about 100 am, about 200 am, about 300 m, about400 am, about 500 am, or more than about 500 am. In some cases, the SNAPmay have a diameter between about 100 nm and 500 nm, between about 200nm and about 400 nm, between about 500 nm and about 10 am, or betweenabout 1000 nm and about 10 m.

In some cases the SNAPs may be covalently attached to the solid supportusing a click chemistry. Generally, the term “click chemistry” is usedto describe reactions that are high yielding, wide in scope, create onlybyproducts that can be removed without chromatography, arestereospecific, simple to perform, and can be conducted in easilyremovable or benign solvents (McKay, C., & Finn M. G. (2014) ClickChemistry in Complex Mixtures Bioorthogonal Bioconjugation vol 21, Issue9, pp 1075-1101; M. G. Meldal, M., & Tornøe, C. W. (2008). Cu-CatalyzedAzide-Alkyne Cycloaddition. Chemical Reviews, 108(8), 2952-3015; Lutz,J., & Zarafshani, Z. (2008). Efficient construction of therapeutics,bioconjugates, biomaterials and bioactive surfaces using azide-alkyne“click” chemistry. Advanced Drug Delivery Reviews, 60(9), 958-970.,herein incorporated by reference).

In some cases, the click chemistry reaction may be a CuAAC, SPAAC,SPANC, or as described elsewhere herein. In some cases, the clickchemistry reaction may need a copper source such as, for example, CuSO₄,Cu(0), CuBr(Ph₃P)₃, CuBr, CuBr/Cu(OAc)₂, CuBr₂, [Cu(CH3CN)4]PF6,PS—NMe2:CuI, silica:CuI, (EtO)3P:CuI, CuCl/Pd2(dba)3, CuBF4, CuCl,CuCl2, Cu(AcO)2, Cu(2), TTA:CuSO4, Cu(1) zeolite (USY), Cu(CH3CN)4OTf,CuOTf, Cu(2):bis-batho, or a combination thereof. In some cases a coppersource is not needed for the click chemistry reaction to proceed. Insome cases, the reducing agent of the click chemistry reaction may be,for example, NaAsc, air, ICl, oxygen, N₂, HAsc, TCEP, dithithreitol(DTT), PPh₃, mercaptoethanol, tris(2-carboxyethyl)phosphine (TCEP),TCEPT-hydrochloric acid a combination thereof, or no reducing agent. Insome cases, the solvent of the click chemistry reaction may be, forexample, THF, pyridine, DMSO, DMF, toluene, NMP, acetonitrile, water,tBuOH, iBuOH, EtOH, MeOH, dioxane, dichloromethane, HEPES, NaCl buffer,acetone, PBS, SFM, Tris buffer, borate buffer, PB, TFH, AcOEt, PIPES,urea, acetone, Tris, saline, AllOCO₂Me, TMS-N₃, urea solution,bicarbonate buffer, a combination thereof, or no solution. In somecases, the base of the click chemistry reaction may be, for example,DIPEA, Lut Na2CO3, iPr₂NH, DBU, Et₃N, Et₃N HCl, Et₃NH+-OAc, K₂CO₃, TBAF,CuSO₄, PS—NMe₂, piperidine, a desired pH, or a combination thereof. Insome cases, the ligand of the click chemistry reaction may be, forexample, TBTA, proline, BMAH, Lut, chiral Lig's, pyridine, His, Batho,TTA, Bim, Phen, Bipy, PMDETA, dNbipy, TRMEDA, or a combination thereof.In some cases, the temperature of the click chemistry reaction may be,for example, 0-5° C., 5-15° C., 15-25° C., 20-25° C., 25-35° C., 35-45°C., 45-55° C., 55-65° C., 65-75° C., 75-85° C., 85-95° C., or greater.In some cases, the temperature of the click chemistry reaction may beless than 0° C. In some reactions, the click chemistry reaction may becovered by aluminum foil. In some cases, the click chemistry reactionmay include an acid, for example, trifluoroacetic acid, trichloroaceticacid, or tribromoacetic acid.

In some cases, a crosslinker may be used for conjugation. In some cases,the crosslinker may be a zero-length crosslinker, homobifunctionalcrosslinker, heterobifunctional crosslinker, or a trifunctional crosslinker. Crosslinkers may be incorporated into a biomolecule preformed orin-situ.

In some cases, zero-length crosslinkers mediate the conjugation forbioconjugation by forming a bond containing no additional atoms. Thus,one atom of a molecule is covalently attached to an atom of a secondmolecule with no intervening linker or spacer. In so conjugationschemes, the final complex is bound together by virtue of chemicalcomponents that add foreign structures to the substances beingcrosslinked. Carbodiimides may be used to mediate the formation of amidelinkages between carboxylates and amines or phosphoramidate linkagesbetween phosphates and amines and are popular type of zero-lengthcrosslinker that may be used, being efficient in forming conjugatesbetween two protein molecules, between a peptide and a protein, betweenan oligonucleotide and a protein, between a biomolecule and a surface orparticle, or any combination of these with small molecules. In somecases, EDC (or EDAC; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride) may be used for conjugating biomolecules containingcarboxylates and amines. In some cases, CMC, or1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (usually synthesized asthe metho p-toluene sulfonate salt), is a water soluble reagent used toform amide bonds between one molecule containing a carboxylate and asecond molecule containing an amine that may be used as a crosslinkerfor bioconjugation. In some cases, DIC, or diisopropyl carbodiimide maybe used for bioconjugation as a zero-length crosslinker. In some cases,DCC (dicyclohexyl carbodiimide) may be used for bioconjugation as azero-length crosslinker. In some cases, Woodward's reagent K isN-ethyl-3-phenylisoxazolium-3′-sulfonate, a zero-length crosslinkingagent able to cause the condensation of carboxylates and amines to formamide bonds. In some cases, CDI, or N,N′-carbonyl diimidazole may beused for bioconjugation as a zero-length crosslinker. In some cases,schiff base formation and reductive amination may be used forbioconjugation as a zero-length cross linker.

In some cases, homobifuctional crosslinkers mediate the conjugation forbioconjugation. In some cases, homofictuional NHS esters may be used forbioconjugation. For example, Lomant's reagent[(dithiobis(succinimidylpropionate), or DSP]) is a homobifunctional NHSester crosslinking agent containing an eight-atom spacer 12 Å in length.The sulfo-NHS version of DSP, dithiobis(sulfosuccin-imidylpropionate) orDTSSP, is a water soluble analog of Lomant's reagent that can be addeddirectly to aqueous reactions without prior organic solvent dissolution.In some cases, disuccinimidyl suberate (DSS), an amine-reactive,homobifunctional, NHS ester, crosslinking reagent produces an eight-atombridge (11.4 Å) between conjugated biomolecules. In some cases,disuccinimidyl tartarate (DST), a homobifunctional NHS estercrosslinking reagent that contains a central diol that is susceptible tocleavage with sodium periodate may be used forms amide linkages withα-amines and F-amines of proteins or other amine-containing molecules.In some cases, BSOCOES [bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone], a water-insoluble, homobifunctional NHS ester crosslinkingreagent that contains a central sulfone group, where the two NHS esterends are reactive with amine groups in proteins and other molecules toform stable amide linkages. In some cases, ethyleneglycolbis(succinimidylsuccinate) (EGS), a homobifunctional crosslinkingagent that contains NHS ester groups on both ends. The two NHS estersare amine reactive, forming stable amide bonds between cross-linkedmolecules within a pH range of about 7 to 9. In some cases,disuccinimidyl glutarate (DSG), a water-insoluble, homobifunctionalcrosslinker containing amine-reactive NHS esters at both ends, may beused for biconjugation. In some cases, N,N′-Disuccinimidyl carbonate(DSC), the smallest homobifunctional NHS ester crosslinking reagentavailable may be used. In some cases, Dimethyl adipimidate (DMA),Dimethyl pimelimidate (DMP), Dimethyl suberimidate (DMS), dimethyl3,3′-dithiobispropionimidate (DTBP),1,4-di-[3′-(2′-pyridyldithio)propionamido] butane, bismaleimidohexane,1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene,DFDNPS (4,4′-difluoro-3,3′-dinitrophenylsulfone),Bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde,Glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic dihydrazide,carbohydrazide, 3,3′-dimethylbenzidine, p-diaminodiphenyl, or haloacetylderivatives may be used as homobifunctional crosslinkers.

In some cases, heterobifuctional crosslinkers mediate the conjugationfor bioconjugation. Heterobifunctional reagents can be used to crosslinkproteins and other molecules in a two- or three-step process. In somecases, one protein is modified with a heterobifunctional compound usingthe crosslinker's most reactive or most labile end. The modified proteinmay then be purified from excess reagent by gel filtration or rapiddialysis. In some cases, heterobifunctionals contain at least onereactive group that displays extended stability in aqueous environments,therefore allowing purification of an activated intermediate beforeadding the second molecule to be conjugated. For instance, anN-hydroxysuccinimide (NHS ester-aleimide hetero-bifunctional can be usedto react with the amine groups of one protein through its NHS ester end(the most labile functionality), while preserving the activity of itsmaleimide functionality. Since the maleimide group has greater stabilityin aqueous solution than the NHS ester group, a maleimide-activatedintermediate may be created. After a quick purification step, themaleimide end of the crosslinker can then be used to conjugate to asulfhydryl-containing molecule. Heterobifunctional crosslinking reagentsmay also be used to site-direct a conjugation reaction toward particularparts of target molecules. In some cases, amines may be coupled on onemolecule while sulfhydryls or carbohydrates are targeted on anothermolecule. In some cases, heterobifunctional reagents containing onephoto-reactive end may be used to insert nonselectively into targetmolecules by UV irradiation. Another component of heterobifunctionalreagents is the cross-bridge or spacer that ties the two reactive endstogether. Crosslinkers may be selected based not only on theirreactivities, but also on the length and type of cross-bridge theypossess. Some heterobifunctional families differ solely in the length oftheir spacer. The nature of the cross-bridge may also govern the overallhydrophilicity of the reagent. For instance, polyethylene glycol(PEG)-based cross-bridges create hydrophilic reagents that provide watersolubility to the entire heterobifunctional compound. In some cases, anumber of heterobifunctionals contain cleavable groups within theircross-bridges, lending greater flexibility to the experimental design. Afew crosslinkers contain peculiar cross-bridge constituents thatactually affect the reactivity of their functional groups. For instance,it is known that a maleimide group that has an aromatic ring immediatelynext to it is less stable to ring opening and loss of activity than amaleimide that has an aliphatic ring adjacent to it. In addition,conjugates destined for use in vivo may have different propertiesdepending on the type of spacer on the associated crosslinker. Somespacers may be immunogenic and cause specific antibody production tooccur against them. In other instances, the half-life of a conjugate invivo may be altered by the choice of cross-bridge, especially when usingcleavable reagents. In some cases, the heterobifunctional crosslinkermay be N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), standardSPDP, LC-SPDP, sulfo-LC-SPDP,succinimidyloxycarbonyl-α-methyl-α-(2-pyri-dyldithio) toluene,succinimidyl-4-(N-maleimidomethyl)cyclo-hexane-1-carboxylate,sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,m-maleimidobenzoyl-N-hydroxysuccinimide ester,m-maleimidobenzoyl-N-hydroxysulfo-succinimide ester,N-succinimidyl(4-iodoacetyl)aminobenzoate,sulfosuccinimidyl(4-iodoacetyl)amino-benzoate,succinimidyl-4-(p-maleimidophenyl)butyrate,N-(γ-maleimidobutyryloxy)succinimide ester,succinimidyl-3-(bromoacetamide)propionate, succinimidyl iodoacetate,4-(4-N-maleimidophenyl)butyric acid hydrazide,4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide,3-(2-pyridyldithio)propionyl hydrazide,N-hydroxysuccinimidyl-4-azidosalicylic acid,sulfosuccinimidyl-2-(p-azidosalicylamido) ethyl-1,3′-dithiopropionate,N-hydroxysulfosuccinimidyl-4-azido-benzoate,N-succinimidyl-6-(4′-azido-2′-nitropheny-lamino)hexanoate,sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate,N-5-Azido-2-nitrobenzoyloxysuccinimide,Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate,N-succinimidyl-(4-azidophenyl)1,3′-dithiopropionate, sulfosuccinimidyl4-(p-azidophenyl) butyrate, Sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate,sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate, p-Nitrophenyldiazopyruvate, p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate,1-(p-azidosalicylamido)-4-(iodoacetamido)butane,N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio) propionamide,Benzophenone-4-maleimide, p-azidobenzoyl hydrazide,4-(p-azidosalicylamido)butylamine, or p-azidophenyl glyoxal.

Other examples of crosslinkers, but not limited to, may beNHS-PEG₄-Azide, NHS-phosphine, N-7-maleimidobutyryl-oxysulfosuccinimideester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl3-(2-pyridyldithio)propionate), sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, m-maleimidobenzoyl-N-hydroxysuccinimideester, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride,dimethyl pimelimidate, sulfosuccinimidyl6-(3′-(2-pyridyldithio)propionamido)hexanoate,6-(3′-[2-pyridyldithio]-propionamido)hexanoate,tris-(succinimidyl)aminotriacetate, Sulfo-NHS-LC-Diazirine,bismaleimidohexane, 1,4-bismaleimidobutane, sulfosuccinimidyl4-(N-maleimidophenyl)butyrate, Sulfo-SBED Biotin Label Transfer Reagent,succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate, succinimidyl3-(2-pyridyldithio)propionate, sulfosuccinimidyl6-(3′-(2-pyridyldithio)propionamido)hexanoate, L-Photo-Leucine,L-Photo-Methionine, sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, Pierce BS(PEG)5,sulfosuccinimidyl 2-((4,4′-azipentanamido)ethyl)-1,3′-dithiopropionate,Sulfo-NHS—SS-Diazirine, Pierce SM(PEG)n, NHS-dPEG-Mal,N-hydroxysulfosuccinimide, sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate,Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide Hydrochloride,N-α-maleimidoacet-oxysuccinimide ester, Sulfo-NHS-LC-Biotin,bis(sulfosuccinimidyl)suberate,trans-4-(maleimidylmethyl)cyclohexane-1-Carboxylate, bismaleimidohexane,1,8-bismaleimido-diethyleneglycol, N-β-maleimidopropionic acidhydrazide, N-succinimidyl 3-(2-pyridyldithio)-propionate,sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,3-(2-pyridyldithio)propionyl hydrazide, 4-(4-N-maleimidophenyl)butyricacid hydrazide, 3,3′-dithiobis(sulfosuccinimidyl propionate,bis(sulfosuccinimidyl) 2,2,4,4-glutarate-d4, orSuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

In some cases, the alkyne derivative attached to the solid support orSNAP may be, for example, dibenzocyclooctyne-amine,dibenzocyclooctyne-acid, dibenzocyclooctyne-N-hydroxysuccinimidyl ester,dibenzocyclooctyne-N-hydroxysuccinimidyl ester,dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester,ibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester,Dibenzocyclooctyne-S—S—N-hydroxysuccinimidyl ester,dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester,dibenzocyclooctyne-PEG4-acid, dibenzocyclooctyne-maleimide,sulfo-dibenzocyclooctyne-biotin conjugate,(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate,(1R,8S,9s)-Bicyclo6.1.0non-4-yn-9-ylmethanol, APN-BCN,(1R,8S,9s)-Bicyclo6.1.0non-4-yn-9-ylmethanol, ethyl(1R,8S,9s)-bicyclo6.1.0non-4-ene-9-carboxylate, Alkyne-PEG5-acid,(R)-3-Amino-5-hexynoic acid hydrochloride, (S)-3-Amino-5-hexynoic acidhydrochloride, (R)-3-(Boc-amino)-5-hexynoic acid,(S)-3-(Boc-amino)-5-hexynoic acid, N-Boc-4-pentyne-1-amine,4-pentyne-1-amine, Boc-propargyl-Gly-OH, 3-Ethynylaniline,4-Ethynylaniline, PC biotin-alkyne, Propargyl chloroformate,Propargyl-N-hydroxysuccinimidyl ester, N—Z-4-pentyne-1-amine,1-Azido-2-(2-(2-ethoxyethoxy)ethoxy)ethane,0-(2-Azidoethyl)heptaethylene glycol, Click-iT® DIBO-Alexa Fluor® 488,Click-iT® DIBO-Alexa Fluor® 555, Click-iT® DIBO-Alexa Fluor® 594,Click-iT® DIBO-Alexa Fluor® 647, Click-iT® DIBO TAMRA, Click-iT®DIBO-biotin, Click-iT® DIBO-amine, Click-iT® DIBO-maleimide, Click-iT®DIBO-succinimidyl ester, Alexa Fluor® 488 alkyne, Alexa Fluor® 555alkyne, triethylammonium salt, Alexa Fluor® 594 carboxamido-(5-(and6-)propargyl), bis(triethylammonium salt, 3-propargyloxypropanoic acid,succinimidyl ester, biotin alkyne, tetraacetyl fucose alkyne, OregonGreen® 488 alkyne *6-isomer*, iodoacetamide alkyne, or5-carboxytetramethylrhodamine propargylamide.

In some cases, the azide derivative attached to a solid support, SNAP,or biomolecule may be, for example, (S)-5-Azido-2-(Fmoc-amino)pentanoicacid, (S)-(−)-2-Azido-6-(Boc-amino)hexanoic acid (dicyclohexylammonium),(S)-2-Azido-3-(4-tert-butoxyphenyl)propionic acid cyclohexylammoniumsalt, L-Azidohomoalanine hydrochloride, (S)-2Azido-3-(3-indolyl)propionic acid cyclohexylammonium salt,(S)-2-Azido-3-methylbutyric acid cyclohexylammonium salt,(S)-2-Azido-3-phenylpropionic acid (dicyclohexylammonium) salt,Boc-3-azido-Ala-OH (dicyclohexylammonium) salt,N-Boc-4-azido-L-homoalanine (dicyclohexylammonium) salt,N-Boc-6-azido-L-norleucine (dicyclohexylammonium) salt,Boc-4-azido-Phe-OH, (S)-(−)-4-tert-Butyl hydrogen 2-azidosuccinate(dicyclohexylammonium) salt,N2-[(1,1-Dimethylethoxy)carbonyl]-N6-[(2-propynyloxy)carbonyl]-L-lysine,Fmoc-β-azido-Ala-OH, 2-Acetamido-2-deoxy-β-D-glucopyranosyl azide,2-Acetamido-2-deoxy-β-D-glucopyranosyl azide 3,4,6-triacetate,2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl azide,N-Azidoacetylgalactosamine-tetraacylated, N-Azidoacetylglucosamine,N-Azidoacetylglucosamine-tetraacylated,6-Azido-6-deoxy-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose,1-Azido-1-deoxy-β-D-galactopyranoside,1-Azido-1-deoxy-β-D-galactopyranoside tetraacetate,6-Azido-6-deoxy-D-galactose, 1-Azido-1-deoxy-β-D-glucopyranoside,2-Azido-2-deoxy-D-glucose, 6-Azido-6-deoxy-D-glucose,1-Azido-1-deoxy-β-D-lactopyranoside,3-Azido-2,3-dideoxy-1-O-(tert-butyldimethylsilyl)-3-D-arabino-hexopyranose,2-Azido-D-galactose tetraacetate,1,2-Di-O-acetyl-3-azido-3-deoxy-5-β-(p-toluoyl)-D-ribofuranose,α-D-Mannopyranosyl azide tetraacetate,2,3,4,6-Tetra-O-acetyl-1-azido-1-deoxy-α-D-galactopyranosyl cyanide,2,3,4-Tri-O-acetyl-β-D-xylopyranosyl azide, 3′-Azido-3′-deoxythymidine,γ-(2-Azidoethyl)-ATP sodium salt solution, γ-[(6-Azidohexyl)-imido]-ATPsodium salt, (2'S)-2′-Deoxy-2′-fluoro-5-ethynyluridine,5-Ethynyl-2′-deoxycytidine, N6-Propargyl-ATP sodium salt,4-Acetamidobenzenesulfonyl azide,(E)-N-(2-Aminoethyl)-4-{2-[4-(3-azidopropoxy)phenyl]diazenyl}benzamidehydrochloride, Azidoacetic acid NHS ester, 1-Azidoadamantane,4-Azidoaniline hydrochloride,(4S)-4-[(1R)-2-Azido-1-(benzyloxy)ethyl]-2,2-dimethyl-1,3-dioxolane,NHS-PEG₄-azide,[3aS-(3aα,4α,5β,7aα)]-5-Azido-7-bromo-3a,4,5,7α-tetrahydro-2,2-dimethyl-1,3-benzodioxol-4-ol,3′-Azido-3′-2-azido-1-methylquinolinium tetrafluoroborate,5-Azidopentanoic acid, 4-Azidophenacyl bromide, 4-Azidophenylisothiocyanate, 3-(4-Azidophenyl)propionic acid, 3-Azido-1-propanamine,3-Azido-1-propanol, Azo biotin-azide, Biotin picolyl azide, tert-Butyl2-(4-{[4-(3-azidopropoxy)phenyl]azo}benzamido)ethylcarbamate,4-Carboxybenzenesulfonazide, 7-(Diethylamino)coumarin-3-carbonyl azide,Ethidium bromide monoazide, Ethyl azidoacetate,4-Methoxybenzyloxycarbonyl azide, aryl azides, diazierines, orO-(2-Aminoethyl)-O′-(2-azidoethyl)heptaethylene glycol,bromoacetomido-PEG₃-azide, iodoacetamide-azide, Alexa Fluor® 488 azide,Alexa Fluor® 488 5-carboxamido-(6-azidohexanyl), bis(triethylammoniumsalt), Alexa Fluor® 555 azide triethylammonium salt, Alexa Fluor® 594carboxamido-(6-azidohexanyl), bis(triethylammonium salt), Alexa Fluor®647 azide triethylammonium salt, 3-(azidotetra(ethyleneoxy))propionicacid succinimidyl ester, biotin azide, L-azidohomoalanine,L-homopropargylglycine, Click-iT® farnesyl alcohol azide,15-azidopentadecanoic acid, 12-azidododecanoic acid, tetraacetylatedN-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine,tetraacetylated N-azidoacetylglucosamine, iodoacetamide azide, ortetramethylrhodamine 5-carboxamido-(6-azidohexanyl).

In some cases, the SNAPs may be covalently attached to the solid supportusing an inherent chemistry of the SNAP. In some cases, the solidsupport may be covered with functional groups that may be reactive tothe SNAP. These functional groups, for example, may be hydroxyl,carbonyl, carboxyl, amino, amides, azides, alkynes, alkenes, phosphates,sulfhydryl, thiols, isothiocyanates, isocyanates, acyl azides, NHSesters, silane, sulfonyl chlorides, aldehydes, esters, glyoxals,epoxides, oxiranes, alkanethiols, carbonates, aryl halides, imidoesters,carbodiimides, anhydrides, fluorophenyl esters, amines, thymines or acombination thereof. In some cases, the SNAP may have a functional groupthat may react with a functional group on the solid support to form acovalent bond. For example, a DNA SNAP may be attached to a solidsupport by reacting one or more thymines in the DNA with amines on thesolid support. For example, the —NH₂ at the N-terminus of a polypeptidechain or —COOH at the C-terminus of a polypeptide chain may react withan appropriate functional group and be attached to the solid supportthrough a covalent bond. In some cases, for example, the functionalgroup of a SNAP may be hydroxyl, carbonyl, carboxyl, amino, amides,azides, alkynes, silane, alkenes, phosphates, sulfhydryl, thiols,isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonylchlorides, aldehydes, esters, glyoxals, epoxides, oxiranes,alkanethiols, carbonates, aryl halides, imidoesters, carbodiimides,anhydrides, fluorophenyl esters, amines, thymines or a combinationthereof. Other bioconjugation processes, reactions, and functionalgroups are described elsewhere within that may be used to attach a SNAPto a solid support. Such a reaction could be spontaneous, or could beinduced by application of heat or ultraviolet radiation.

In some cases, silane chemistry may be employed for bioconjugation. Insome cases, functional silane compounds containing an organofunctionalor organo-reactive arm can be used to conjugate biomolecules toinorganic substrates. The appropriate selection of the functional orreactive group for a particular application can allow the attachment ofproteins, oligonucleotides, whole cells, organelles, or even tissuesections to substrates. The organosilanes used for these applicationsmay include functional or reactive groups such as hydroxyl, amino,aldehyde, epoxy, carboxylate, thiol, and even alkyl groups to bindmolecules through hydrophobic interactions. In some cases,3-Aminopropyltriethoxysilane (APTS) and 3-Aminopropyltrimethoxysilaneare used to create a functional group on an inorganic surface orparticle. In some cases, once deposited on a substrate, the alkoxygroups form a covalent polymer coating with the primary amine groupssticking off the surface and available for subsequent conjugation.Carboxyl- or aldehyde-containing ligands may be directly coupled to theaminopropyl groups using a carbodiimide reaction or reductive amination.In some cases, alternatively, surfaces initially derivatized with anaminopropylsilane compound can be modified further with spacer arms orcrosslinkers to create reactive groups for coupling affinity ligands orbiomolecules. For instance, the amine groups may be derivatized with anNHS-PEGn-azide compound for use in click chemistry or Staudingerligation reactions for linking proteins or other biomolecules. In somecases, APTS-modified surfaces may be further derivatized withamine-reactive crosslinkers to create additional surface characteristicsand reactivity. Modification with NHS-PEG4-azide forms a hydrophilic PEGspacer terminating in an azido group that can be used in a clickchemistry or Staudinger ligation reaction to couple other molecules.

In some cases, other crosslinking agents that contain an amine-reactivegroup on one end also may be used to modify and activate theAPTS-modified substrate. Surfaces may be designed to contain, forinstance, reactive hydrazine or aminooxy groups for conjugation withcarbonyl-containing molecules, such as aldehydes formed throughperiodate oxidation of carbohydrates or natively present at the reducingend of sugars and glycans.

In some cases, the amine groups on ATPS surfaces may be acylated usingglutaric anhydride to create carboxylate functionalities, which werethen activated with NHS/DCC to form the NHS ester. This derivative couldbe used to couple amine-containing proteins and other molecules viaamide bond formation. In a second activation strategy, the aminopropylgroups on the surface were activated with 1,4-phenylenediisothiocyanate(PDITC) to create terminal isothiocyanate groups for coupling amines.Both methods resulted in the successful coupling of amine-dendrimers tosilica surfaces for use in arrays. In some cases, amine surfacesprepared using an aminosilane compound can be modified to containcarboxylate groups using the following protocol involving the reactionwith an anhydride, such as succinic anhydride or glutaric anhydride.After modification, the carboxylates then can be used to coupleamine-containing molecules using a carbodiimide reaction with EDC plussulfo-NHS. In some cases, modification of an APTS surface with glutaricanhydride creates terminal carboxylates for coupling of amine-containingligands which may be used for bioconjugation.

In some cases, aminosilane surfaces also may be activated by use of abifunctional crosslinker to contain reactive groups for subsequentcoupling to biomolecules. In one such reaction, N,N′-disuccinimidylcarbonate (DSC) was used to react with the amines on a slide surface andcreate terminal NHS-carbonate groups, which then could be coupled toamine-containing molecules, which may be used for bioconjugation. Insome cases, APTS-modified surfaces can be activated with DSC to formamine-reactive succinimidyl carbonates for coupling proteins or otheramine-containing molecules.

In some cases, silane coupling agents containing carboxylate groups maybe used to functionalize a surface with carboxylic acids for subsequentconjugation with amine-containing molecules. For example,carboxyethylsilanetriol contains an acetate organo group on asilanetriol inorganic reactive end. The silanetriol component isreactive immediately with inorganic —OH substrates without priorhydrolysis of alkoxy groups, as in the case with most other silanizationreagents. In some cases, carboxyethylsilanetriol has been used to addcarboxylate groups to fluorescent silica nanoparticles to coupleantibodies for multiplexed bacteria monitoring. This reagent can be usedin similar fashion to add carboxylate functionality to many inorganic ormetallic nano-materials, which also will create negative chargerepulsion to maintain particle dispersion in aqueous solutions. In somecases, covalent coupling to the carboxylated surface then can be done byactivation of the carboxylic acid groups with a carbodiimide tofacilitate direct reaction with amine-containing molecules or to formintermediate NHS esters, which may be used for bioconjugation. In somecases, carboxylethylsilanetriol can be used to modify an inorganicsubstrate to containing carboxylate groups for coupling amine-containingligands.

In some cases, silane modification agents such as glycidoxy may beutilized for bioconjugation to a surface substrate. Glycidoxy compoundscontain reactive epoxy groups. Surfaces covalently coated with thesesilane coupling agents can be used to conjugate thiol-, amine-, orhydroxyl-containing ligands, depending on the pH of the reaction. Insome cases, 3-glycidoxy-propyltrimethoxysilane (GOPTS) or3-glycidoxypro-pyltriethoxysilane can be used to link inorganic silicaor other metallic surfaces containing —OH groups with biologicalmolecules containing any three of these major functional groups. In somecases, epoxy-containing silane coupling agents form reactive surfacesthat can be used to couple amine-, thiol-, or hydroxyl-containingligands which may be used for bioconjugation.

In some cases, the reaction of the epoxide with a thiol group yields athioether linkage, whereas reaction with a hydroxyl gives an ether andreaction with an amine results in a secondary amine bond. The relativereactivity of an epoxy group is thiol>amine>hydroxyl, and this isreflected by the optimal pH range for each reaction. In this case, thelower the reactivity of the functional group the higher the pH requiredto drive the reaction efficiently.

In some cases, isocyanates groups may be utilized for bioconjugation toa surface support. Isocyanate groups are extremely reactive towardnucleophiles and will hydrolyze rapidly in aqueous solution which areespecially useful for covalent coupling to hydroxyl groups undernonaqueous conditions, which is appropriate for conjugation to manycarbohydrate ligands. Silanization can be accomplished in dry organicsolvent to form reactive surfaces while preserving the activity of theisocyanates. Isocyanatopropyltriethoxysilane (ICPTES) contains anisocyanate group at the end of a short propyl spacer, which is connectedto the triethoxysilane group useful for attachment to inorganicsubstrates. In some cases, the isocyanate-containing silane couplingmagnet can be used to couple hydroxyl-containing molecules to inorganicsurfaces which may be used for bioconjugation.

In some cases, ICPTES may be used to create novel chitosan-siloxanehybrid polymers by coupling the isocyanate groups to the functionalgroups of the carbohydrate and forming a silica polymer using thetriethoxysilane backbone. In some cases, ICPTES and APTS have been usedin combination to create organically modified silica xerogels throughcarboxylic acid solvolysis that formed hybrid materials with luminescentproperties.

In some cases, nanoparticles or microparticles may be utilized as asurface support for bioconjugation. In some cases, particle types andcompositions of almost limitless shape and size, including spherical,amorphous, or aggregate particles, as well as elaborate geometric shapeslike rods, tubes, cubes, triangles, and cones. In addition, newsymmetrical organic constructs have emerged in the nanometer range thatinclude fullerenes (e.g., Bucky-balls), carbon nanotubes, anddendrimers, which are highly defined synthetic structures used asbioconjugation scaffolds. The chemical composition of particles may bejust as varied as their shape. Particles can comprise of polymers orcopolymers, inorganic constructs, metals, semiconductors,superparamagnetic composites, biodegradable constructs, syntheticdendrimers, and dendrons. Polymeric particles can be constructed from anumber of different monomers or copolymer combinations. Some of the morecommon ones include polystyrene (traditional “latex” particles),poly(styrene/divinylbenzene) copolymers, poly(styrene/acrylate)copolymers, polymethylmethacrylate (PMMA), poly (hydroxyethylmethacrylate) (pHEMA), poly (vinyltoluene), poly(styrene/butadiene)copolymers, and poly(styrene/vinyltoluene) copolymers. In some cases, bymixing into the polymerization reaction combinations of functionalmonomers, one can create reactive or functional groups on the particlesurface for subsequent coupling to affinity ligands. One example of thisis a poly(styrene/acrylate) copolymer particle, which createscarboxylate groups within the polymer structure, the number of which isdependent on the ratio of monomers used in the polymerization process.In some cases, inorganic particles are used extensively in variousbioapplications. For example, gold nanoparticles may be used fordetection labels for immunohistochemical (IHC) staining and lateral flowdiagnostic testing. In some cases, the use of particles inbioapplications like bioconjugation involves the attachment of affinitycapture ligands to their surface, by either passive adsorption orcovalent coupling. The coupling of an affinity ligand to such particlescreates the ability to bind selectively biological targets in complexsample mixtures. The affinity particle complexes can thus be used toseparate and isolate proteins or other biomolecules or to specificallydetect the presence of these targets in cells, tissue sections, lysates,or other complex biological samples. In some cases, the reactions usedfor coupling affinity ligands to nanoparticles or microparticles arebasically the same as those used for bioconjugation of moleculesdescribed herein.

In some cases, particle type used for bioapplications (e.g.bioconjugation) is the polymeric microsphere or nano-sphere, whichcomprises a spherical, nonporous, “hard” particle made up of long,entwined linear or crosslinked polymers. In some cases, creation ofthese particles involves an emulsion polymerization process that usesvinyl monomers, sometimes in the presence of divinyl crosslinkingmonomers. In some cases, larger microparticles may be built fromsuccessive polymerization steps through growth of much smallernanoparticle seeds. In some cases, polymeric particles comprise ofpolystyrene or copolymers of styrene, like styrene/divinylbenzene,styrene/butadiene, sty-rene/acrylate, or styrene/vinyltoluene. Othercommon polymer supports include polymethylmethacrylate (PMMA),polyvinyltoluene, poly(hydroxyethyl meth-acrylate) (pHEMA), and thecopolymer poly(ethylene glycol dimethacrylate 2-hydroxyethylmetacrylate)[poly(EGDMA/HEMA)].

In some cases, one method of attaching biomolecules to hydrophobicpolymeric particles is the use of passive adsorption. In some cases,protein adsorption onto hydrophobic particles takes place through stronginteractions of nonpolar or aromatic amino acid residues with thesurface polymer chains on the particles with concomitant exclusion ofwater molecules. Since proteins usually contain hydrophobic corestructures with predominately hydrophilic surfaces, their interactionwith hydrophobic particles must involve significant conformationalchanges to create large-scale hydrophobic contacts.

In some cases, particle types contain functional groups that are builtinto the polymer backbone and displayed on their surface. The quantityof these groups can vary widely depending on the type and ratios ofmonomers used in the polymerization process or the degree of secondarysurface modifications that have been performed. In some cases,functionalized particles can be used to couple covalently biomoleculesthrough the appropriate reaction conditions.

Common Functional Groups or Reactive Groups on Particles forBioconjugation

In some cases, a particle may couple with a crosslinker forbioconjugation.

In some cases, the rate of attachment of DNA SNAPs s to the solidsupport, or the efficacy or strength of attachment, may be altered byaltering the sequence of DNA comprising the SNAP. For example, in thecase of a DNA SNAP attached to a solid support by a reaction involvingone or more thymines the attachment may be varied by varying the numberof thymines in the DNA sequence. In some cases, increasing the number ofthymines may facilitate the attachment of the SNAP to the solid support.

In some cases, the solid support is a part of a flow cell. In somecases, the SNAPs may be attached to a solid support in a flow cell. Insome cases, the SNAPs may be directly conjugated to a solid support in aflow cell. In some cases, the SNAPs may be adsorbed to a solid supportin a flow cell. Attaching the SNAPs in the flow cell may allowvisualization of the SNAPs as they attach to the solid support. Theattachment of the SNAPs may be optimized by monitoring the number ofattached SNAPs compared to the number of attachment sites during theattachment process. In some cases, the attachment of the SNAPs may beoptimized by monitoring the area of the solid support covered by theSNAPs and the area of the solid support that is unoccupied by the SNAPsduring the attachment process.

In some cases, the SNAPs may be conjugated directly in a flow cell. Insome cases, the SNAPs may be conjugated to a surface within the flowcell. In some cases, the SNAPs may be conjugated to a surface within theflow cell before being conjugated to the biological or chemicalentities. In some cases, a biological or chemical entity may be flowedinto a flow cell and conjugated to a SNAP that is already conjugated tothe solid support. In some cases, a biological or chemical entity may beconjugated to a SNAP before the SNAP is introduced into a flow cell andconjugated to a solid support in a flow cell. In some cases, abiological or chemical entity and a SNAP may be introduced into a flowcell and conjugated to each other within the flow cell, before the SNAPis conjugated to a solid support within the flow cell.

In some cases, the biological or chemical entities may be conjugated tothe SNAPs prior to attaching the SNAPs to a solid support. Afterperforming such a reaction the products may be purified to separate outconjugated SNAP-biological/chemical entity moieties from unconjugatedSNAPs and biological/chemical entities.

The methods of this disclosure may be used to spatially separatebiological or chemical entities. In some embodiments, methods of thisdisclosure may be used to spatially separate proteins, small molecules,DNAs, RNAs, glycoproteins, metabolites, carbohydrates, enzymes, orantibodies. In some embodiments, methods of this disclosure may be usedto spatially separate complexes, such as protein complexes comprisingtwo or more proteins, protein nucleic acid complexes, or othercomplexes. In some cases, the methods may be used to spatially separateviral particles or viroids. In some cases, the methods may be used toseparate cells, such as bacterial cells, microbial cells, mammaliancells or other cells.

In some embodiments, the SNAP may be formed on the seed prior to theseed being attached to the biological or chemical entity.

In some embodiments this disclosure provides a composition comprising anucleic acid SNAP attached to a protein, a nucleic acid SNAP attached toa small molecule, a nucleic acid SNAP attached to a protein complex, anucleic acid SNAP attached to a protein nucleic acid SNAP, a nucleicacid SNAP attached to a carbohydrate, a nucleic acid SNAP attached to aviral particle or a nucleic acid SNAP attached to a cell.

In some embodiments this disclosure provides a composition comprising adendrimer attached to a protein, a dendrimer attached to a smallmolecule, a dendrimer attached to a protein complex, a dendrimerattached to a protein dendrimer, a dendrimer attached to a carbohydrate,a dendrimer attached to a viral particle or a dendrimer attached to acell.

In some cases, the biological or chemical entities may be eluted fromthe solid support either by cleaving a photo-cleavable bond, or bychemically or enzymatically digesting the SNAP.

In some cases, the biological or chemical entities may attach to thesolid support directly, while the SNAPs occlude other biological orchemical entities from attaching in the immediate vicinity. In somecases the biological or chemical entities may attach directly to anattachment site within a microwell or nanowell, and the size of theSNAPs may be selected to prevent more than one SNAP from occupying themicrowell or nanowell. In such cases, the SNAP may be removed, either bycleaving a photo-cleavable bond, or by chemically or enzymaticallydigesting the SNAP.

In some embodiments, SNAPs of this disclosure may be used asnanoparticles. For example, SNAPs of this disclosure may be used asnanoparticles for detection or visualization. In some cases, a nucleicacid SNAP may be formed which incorporates modified nucleotides whichcomprise fluorescent moieties. Any fluorescently labeled nucleotideknown in the art may be used in a SNAP of this disclosure. Examples offluorescently labeled nucleotides include, but are not limited to, AlexaFluor™ 555-aha-dCTP, Alexa Fluor™ 555-aha-dUTP, 1 mM in TE buffer, AlexaFluor™ 647 ATP (Adenosine 5′-Triphosphate, Alexa Fluor™ 6472′-(or-3′)-O—(N-(2-Aminoethyl) Urethane), Hexa(Triethylammonium) Salt),Alexa Fluor™ 647-aha-dCTP, Alexa Fluor™ 647-aha-dUTP, 1 mM in TE buffer,BODIPY™ FL ATP (Adenosine 5′-Triphosphate, BODIPY™ FL2′-(or-3′)-O—(N-(2-Aminoethyl)Urethane), Trisodium Salt), 5 mM inbuffer, BODIPY™ FL ATP-7-S, Thioester (Adenosine5′-O-(3-Thiotriphosphate), BODIPY™ FL Thioester, Sodium Salt), BODIPY™FL GDP (Guanosine 5′-Diphosphate, BODIPY™ FL2′-(or-3′)-O—(N-(2-Aminoethyl) Urethane), Bis (Triethylammonium) Salt),ChromaTide™ Alexa Fluor™ 488-5-UTP, ChromaTide™ Alexa Fluor™ 488-5-dUTP,ChromaTide™ Alexa Fluor™ 546-14-UTP, ChromaTide™ Alexa Fluor™546-14-dUTP, ChromaTide™ Alexa Fluor™ 568-5-dUTP, ChromaTide™ AlexaFluor™ 594-5-dUTP, ChromaTide™ Fluorescein-12-dUTP, ChromaTide™ TexasRed™-12-dUTP, Fluorescein-12-dUTP Solution (1 mM),Fluorescein-aha-dUTP—1 mM in TE Buffer, Guanosine5′-O-(3-Thiotriphosphate), BODIPY™ FL Thioester, Sodium Salt (BODIPY™ FLGTP-7-S, Thioester), Guanosine 5′-Triphosphate, BODIPY™ FL2′-(or-3′)-O—(N-(2-Aminoethyl) Urethane), Trisodium Salt (BODIPY™ FLGTP), Guanosine 5′-Triphosphate, BODIPY™ TR2′-(or-3′)-O—(N-(2-Aminoethyl) Urethane), Trisodium Salt (BODIPY™ TRGTP), MANT-ADP (2′-(or-3′)-O—(N-Methylanthraniloyl) Adenosine5′-Diphosphate, Disodium Salt), MANT-ATP(2′-(or-3′)-O—(N-Methylanthraniloyl) Adenosine 5′-Triphosphate,Trisodium Salt), MANT-GDP (2′-(or-3′)-O—(N-Methylanthraniloyl) Guanosine5′-Diphosphate, Disodium Salt), MANT-GMPPNP(2′-(or-3′)-O—(N-Methylanthraniloyl)-β:γ-Imidoguanosine 5′-Triphosphate,and Trisodium Salt), MANT-GTP (2′-(or-3′)-O—(N-Methylanthraniloyl)Guanosine 5′-Triphosphate, Trisodium Salt).

In some cases, a SNAP of this disclosure may be designed such thatprobes may be attached onto the surface of the SNAP. A SNAP withattached probes may be used as a detection reagent. In some cases, aSNAP with attached probes is also labeled with fluorescent moieties toform a fluorescent detection reagent. In some cases, a SNAP withattached probes and fluorescent moieties may provide a high degree ofsignal amplification. The amount of probes on the SNAP may be titratedto achieve a desired degree of sample amplification. In some cases,differently sized SNAPs may be attached to different probes. In somecases, differently colored SNAPs may be attached to different probes. Insome cases a library of different probes may be attached tofluorescently labeled SNAPs such that a first probe is attached to aSNAP which is a different size and/or color from a SNAP each other probeis attached to.

EXAMPLES Example 1—Generation of DNA SNAPs

Oligos were reconstituted in dH20 to a final concentration of 100 uMwith the exception of the extension primer which was reconstituted to500 uM (2.9 mg/ml). The extension primer was conjugated to Deep Red 200nm bead.

Primer 1- (SEQ ID NO: 1) 5′-GCCAGGGTGCGAGGGTTTGTTTCATTGCTTCACGCCCTTACCCTCGCACCCTGGCACGG Primer 2- (SEQ ID NO: 2)5′-TCCCACGGTGGCACCTCGCACCT Primer 3- (SEQ ID NO: 3)5′-CGCACGCTGCCACCCTCGCTTTTGCGAG GGTGGCAGCGT Primer 4- (SEQ ID NO: 4)5′-GCGAGGTGCGAGGTGCCACCGTGGGACC GT Extension Primer- (SEQ ID NO: 5)5′-AAGGGCGTGAAGCAATGA

Amplification of Template

The following submixes were prepared:

Submix 1:

Water 187.5 μL 1M Tris-HCl pH 7.5 12.5 μL Primer 1 [100 uM] 50 μL

Submix 2:

Water 375 μL 1M Tris-HCl pH 7.5 25 μL Primer 2 [100 uM] 50 μL Primer 4[100 uM] 50 μL

Submix 3:

Water 187.5 μL 1M Tris-HCl pH 7.5 12.5 μL Primer 3 [100 uM] 50 μL

Each submix was aliquoted into 100 μL aliquots and incubated asdescribed below:

Thermocycler conditions:

-   -   95° C. 30 seconds    -   Ramp down to 50° C. at 0.1° C./s    -   Hold at 4° C.

Ligation of the Rolling Circle Template

Mix:

-   -   Submix 1 100 μL    -   Submix 2 100 μL    -   Submix 3 100 μL

Ligation:

10X NEB T4 DNA ligase buffer 200 μL Mixed Oligo submix [5 uM] 200 μLWater 1500 μL Mix.Add 100 μL NEB T4 DNA Ligase [400,000 u/ml]

Complete mixture was aliquoted into PCR tubes and incubated at 20° C.for ˜20 hr followed by 65° C. for 10 minutes.

Solutions were pooled and 50 μL of 100 uM stock extension primers wereadded and mixed. Mixture was aliquoted into 100 μL aliquots in PCR tubesand subjected to the following temperature conditions:

Thermocycler conditions:

-   -   70° C. 30 seconds    -   Ramp down to 40° C. at 0.1° C./s    -   Hold at 4° C.

Template can now be stored at −20° C. until ready to use.

Rolling Circle Amplification for Nanoparticle Construction

PCR Mix:

Water 969 μL 10X NEB phi29 buffer 150 μL TCEP [500 mM] 15 μL BSA [100X]15 μL dNTP mix [10 mM] 1.5 μLVortexed to Mix then Added:

Primed Rolling Circle Template 300 μLVortexed to Mix then Added:

NEB phi29 polymerase [10 Ku/ml] 50 μLInverted to mix. Aliquoted reaction mixture into 63 μL aliquots in PCRtubes.

Incubated at

-   -   30° C. for 120 minutes    -   65° C. for 10 minutes        Pooled samples and added 90 ul of 250 mM EDTA.        Centrifuged sample at 12,500 G for 5 minutes at 4° C. Recovered        supernatant and discarded white pellet.

Analysis of Nanoballs

Serial dilutions were performed 1:100 on sample (100 ul)+1:10 dilutionand 2 ul of Sybr Gold (1×) was added. Applied 1 ul spots on aminesurface treated slides and imaged. As seen in FIG. 3 and FIG. 4 DNASNAPs were successfully formed, and a high degree of co-localization wasseen between the DNA SNAPs and the Deep Red balls.

Analysis of Binding

In some cases, SNAPs can be removed from a surface they attach to, suchas a chip or array. Removal of SNAPs can be mediated for example by ahigh amount of acetonitrile, a high concentration of sodium hydroxide,or a high concentration of salt. A high amount of acetonitrile can be afinal percentage of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100% acetonitrile. A high amount of sodium hydroxide can be atleast about 0.1 M, 0.5 M, 1 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M,or 10 M. A high amount of salt can be at least about 0.1 M, 0.5 M, 1 M,2 M, 3 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, or 10 M. The salt used can befor example MgCl₂ or NaCl. In some cases, a chaotropic reagent such asDMSO or formamide can mediate the removal of SNAPs.

Example 2—SNAP Production Using an Epimark Taq Polymerase

In a further example, SNAP production was optimized using an Epimark Taqpolymerase. SNAP templates were prepared according to the methoddescribed in Example 1, paragraphs [0093]-[0098]. The Taq polymerase wasdetermined to have better control over SNAP size than phi29 polymerases.A 500 μl reaction mixture was prepared using an 8683 ng template. Thefollowing reagents were mixed in a 1.5 ml PCR tube:

PCR Mix:

5X reaction buffer  100 μl 10 mM dNTPs   10 μl Template DNA 16.3 μlFluorescent dNTPs    5 μl Nuclease-free water  369 μl Total volume  500μlThe PCR mix was vortexed briefly in the PCR tube. After vortexing, 2.5μl of Epimark Taq Polymerase was added to the PCR mix. The PCR tube wasinverted to mix the polymerase with the other reagents. The PCR mix wasplaced in a thermal cycler. The template DNA was initially denatured at94° C. for 30 seconds. The reaction mixture was amplified for 30 cyclesunder the following thermal conditions:

-   -   Denaturation at 94° C. for 30 seconds    -   Annealing at 53° C. for 60 seconds    -   Extension at 68° C. for 30 seconds        After the final thermal cycle, the SNAPs were held at 68° C. for        5 further minutes. The PCR tube was then cooled to 4° C. and        held until purification.

SNAPs were purified after synthesis. dNTPs were removed via an EDTA spinpurification method. 30 μl of 250 mM EDTA was added to the 500 μl PCRmix in the PCR tube. The PCR tube was centrifuged at 12,500 G for 5minutes at 4° C. The supernatant was saved and the pellet was discarded.SNAP samples were filtered with 0.22 μm filter tubes, then purifiedusing AKTA FPLC using a gradient method. SNAPs were purified via anionexchange chromatography using 1 L each of deionized water and 1M NaClsolution that had been filtered through 0.22 μm filters. Aftercollecting the SNAP-containing fraction, the solution was desalted usingovernight dialysis in a dialysis cassette. SNAPs were concentrated usinga vacuum centrifuge. The solvent was evaporated at 28° C. forapproximately 4 hours until the final volume was less than 500 μl.

Example 3: Purification of SNAPs

A batch of SNAPs can be produced as described herein. Once the SNAPs areproduced, they can be purified. A three step example protocol of howSNAPs can be purified is described below.

Step 1: Anion Exchange Chromatography

Fast protein liquid chromatography (FPLC) anion exchange chromatographywas used to purify SNAPs. Here, a salt gradient was used to separatedifferentially charged DNA molecules (SNAPs) for collection. Threefractions (bottom, middle, top) were collected for analysis.

An example of the anion exchange purification is shown in FIG. 5.Dynamic light scattering was used to measure the hydrodynamic radius ofparticles in a sample prior to purification (left panel). The radii aredistributed between about 0.1 nm and 10,000 nm. During anion exchangepurification, samples present in an identified target peak (middlepanel) were collected. Dynamic light scattering was used to measure thehydrodynamic radius of particles in the purified sample, and the sampleswere found to comprise SNAPs having hydrodynamic radii around about 100nm and around about 1000 nm.

In some cases, size exclusion chromatography can be performed in lieu ofanion exchange chromatography for purification.

Samples from the three channels were imaged (FIG. 6, left) using astandard microscopy protocol, and intensities were quantified (right).SNAPs from the bottom channel displayed a higher intensity than thosefrom the middle and top channels. Thus, in some cases, the size and/orbrightness of the SNAPs can elute in a particular anion exchangefraction.

Step 2: Dialysis

After the anion exchange chromatography was performed, salt was removedby a standard dialysis protocol. Briefly, dialysis is a separationtechnique that can facilitate the removal of small, unwanted compounds(e.g. salt) from macromolecules (e.g. SNAPs) in solution by selectiveand passive diffusion through a semi-permeable membrane. An anionexchange purified sample comprising SNAPs requiring salt removal and abuffer solution were placed on opposite sides of the membrane. SNAPswere retained on the sample side of the membrane, but salt was able topass freely through the membrane. The salt collected on the side of themembrane opposite the SNAPs, thus reducing the concentration of salt inthe sample. In this way, the concentrations of small contaminants suchas salt within the sample were decreased to acceptable or negligiblelevels.

Step 3: Concentration

Using a standard vacuum centrifugation protocol, batches of SNAPs wereconcentrated with minimal loss compared to conventional approaches.

SNAPs can be concentrated to a final concentration of between 1 μM and100 μM. For example, batches of SNAPs produced have had concentrationsof about 63.6 μM, 47.5 μM, 38 μM, and 8.9 μM.

FIG. 7 shows the absorption spectra at 260 nm (A260) traces of differentSNAP batches. The individual batches were produced using varyingfluorescent dNTP types, fluorescent dNTPs from varying vendors, varyingfluorescent dyes, varying Taq polymerases, and/or Taq polymerases usedfrom varying vendors. Each SNAP displays a similar absorption profile.

Example 4: Production of SNAPs of a Desired Size

SNAPs were produced as described herein, and nanoparticle size wasmeasured.

Methods for measuring nanoparticle (e.g. SNAP) size can include dynamiclight scattering, nanoparticle tracking analysis, and microscopytechniques such as transmission electron microscopy (TEM), scanningelectron microscopy (SEM), and atomic force microscopy (AFM).

Dynamic light scattering, which was used herein, can measure a diffusioncoefficient through constructive and destructive interference patternsof an entire population of SNAPs. Nanoparticle tracking analysis canmeasure the diffusion coefficient through particle tracking ofindividual particles if the particles are greater than 30 nm in size.Microscopy techniques including TEM, SEM, and AFM can measure particlesize and allow subsequent image analysis of individual particles withoutrelying on the scattering of light.

Images were taken using a standard imaging protocol, and hydrodynamicradii of the SNAPs were determined to be between 25 nm and 27 nm byanion exchange chromatography. This size range may allow for multipleSNAPs to occupy each feature in some applications. These small SNAPswere observed to co-localize within a single feature, as seen in FIG. 8.In this example, SNAPs were applied to an array and imaged (SNAP 1 onthe bottom panel, SNAP 2 in the middle panel), and co-localization wasdetermined by merging the images (top panel).

In an additional experiment, larger SNAPs were applied to a chipsurface. In this case, the large SNAPs arranged themselves on the chipsurface, thus achieving a “single occupancy of features.” FIG. 9 showsSNAPs having a large hydrodynamic radius applied to a chip havingfeatures. The SNAPs were imaged in the bottom (SNAP1) and middle (SNAP2)images, and these images are overlaid (SNAP 1 and SNAP 2) in the topimage. Significant co-localization was not observed. Thus, when SNAPsare larger in size, they can sit on the features with very littleco-localization. In contrast, when SNAPs are smaller in size, they canco-localize to features in some cases.

To determine an appropriate size range for SNAPs, SNAP occupancy onarray can be measured. This can be measured as brightness vs. dilution.

SNAPs were titrated to dilutions ranging between 10⁻⁵ and 10⁰ andapplied to an array and signals were measured using dynamic lightscattering. For each dilution, the number of counts and number offeatures occupied by a SNAPs was determined (FIG. 10). The dark linesrepresent a fluorescent control, while the light lines represent theSNAPs. The dotted lines represent the number of features occupied, whilethe solid lines represent the number of counts. Different signal trendsfor small molecules were observed for dye vs. SNAPs on the array.

A constant number of counts was recorded for the SNAPs (solid lightline) regardless of dilution factor, which can suggest discreteoccupancy of the SNAPs on features of the array.

The apparent number of occupied features was observed to change withdetection threshold (dotted light line). This can suggest that thenumber of features occupied can be a function of the dilution. Areceiver operating characteristic (ROC) curve can be developed fordetection sensitivity and specificity.

SNAPs produced can be a variety of sizes. In some cases, SNAPs can beabout 1 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90nm, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm in diameter. The diametercan be a major diameter, a minor diameter, or an average diameter. Insome cases, SNAPs can be produced such that SNAPs having a range ofdiameters can be produced.

Example 5: Brightness of SNAPs

A batch of SNAPs were separately applied to an array having features andimaged using a standard imaging protocol. with a maximum grey value ofabout 12,000 and a difference in grey value between areas having and nothaving SNAPs of about 6,000, as measured by the variations in gray valueacross the array. Thus, SNAPs are able to bind to the array, andconjugated SNAPs can and be detected.

Example 6: Measurement of the Concentration of SNAPs

After the SNAPs are concentrated, the concentration of the SNAPs can bemeasured. For example, amine conjugated SNAPs can be quantitated usingan o-phthaldialdehyde (OPA) free amine reaction, as shown in the topleft of FIG. 12. Briefly, OPA can react with the amine to enablefluorescent detection, and can enable quantitation when a standard curve(e.g. PolyT amine serial dilutions between 0 and 100 μM, bottom left ofFIG. 12) is performed.

The fluorescence resulting from such a reaction in three separatebatches of SNAPs was measured at Ex/Em: 380 nm/460 nm, as shown in FIG.12 (right). The A260 (absorbance at 260 nm) was quantified, and appliedto the standard curve to determine the concentration of each batch. Thethree batches were determined to have concentrations of 47.5 μM, 38 μM,and 8.9 μM. Another batch was determined to have a concentration of 63.6μM (data not shown). This assay can measure the concentrations of SNAPsat least in the range of 1 μM and 100 μM. In some cases, a sample ofSNAPs can be diluted as necessary to fit into this range. Using thisassay, relatively small amounts of amine modified DNA (e.g. SNAPs) canbe measured.

Example 7: SNAP Conjugation #1

An experiment was performed such that click conjugation ofAzide-AlexaFluor 586 to a SNAP was carried out on a chip. DBCO-SNAPs(488) (SNAPs having a DBCO group conjugated to a dye that can fluoresceat 488 nm) was immobilized onto an array by flow, and images wereacquired at 488 nm (SNAP channel) and 575 nm (Azide dye channel). Then,Azide-568 (Azide-AlexaFluor 568), which can fluoresce at 568 nm, wasincubated on the array to allow for a conjugation reaction between theDBCO and the Azide, and the array was washed after the incubation.Following this protocol, images were again acquired at 488 nm and 575 nmto assess the extent of the DBCO-Azide reaction. After incubation, theAzide dye channel showed significantly more fluorescence than before.The SNAP channel (control) showed similar signal before and after thereaction (FIG. 14). This shows that the click conjugation between DBCOand Azide on a chip (array) can be feasible.

For both channels, pre- and post-incubation, signal to noise ratio andintensity were measured for the dark (darker shading) and bright(brighter shading) sections (FIG. 14). These data confirm that theintensity is significantly increased after the click reaction, and thatthe click reaction performs well on the chip.

An additional set of SNAPs was then immobilized on arrays and conjugatedto Azide-AlexaFluor 568, wherein the Azide-AlexaFluor 568 was applied at10× excess of the total number of features. There were about 23.5million features per flow channel. Images were taken at 488 nm and 568nm, pre- and post-incubation, and the intensities were quantified. Theintensities at 488 nm were slightly lower at 488 nm, which can be aneffect of differential manual washing of block solution (FIG. 15).Intensities at 568 nm were significantly higher after incubation with10× Azide-AlexaFluor 568 (about 2-2.5 fold), as shown in FIG. 16. Inother words, uniform, localized signal at 568 nm was observed afterAzide-AlexaFluor 568 was conjugated in 10× excess of the number offeatures per channel.

Example 8: SNAP Conjugation #2

An experiment was performed such that click conjugation of PE-conjugated(R-Phycoerythrin-conjugated) azide was carried out on a chip. SNAPs wereprepared with a DBCO handle and a nucleotide capable of fluorescing at488 nm. SNAPs were deposited on a chip surface and allowed to incubatefor 1 hour to attach to the chip surface. The chip was incubated forbetween 15 minutes and 30 minutes with a blocking buffer, and PE (1mg/ml) with Azide handles on Amines was flown over the chip such thatthere was a 1000× molar excess of PE SNAPs. The chip was then incubatedovernight. The channels were then washed with phosphate buffered salinewith 2% tween (PBST) and imaged.

The image is shown in FIG. 17. Overall, there was a high PE signalthroughout the flow channel, which can suggest an apparently highnon-specific binding. A punctate signal, or an on and off feature, wasobserved. The on feature was observed to be typically higher byapproximately 2000 counts. DBCO SNAPs were present on features, andproduced a signal which was low compared with the signal from the PE. Insome cases, the batch of SNAPs can be optimized e.g. by improving thequality and increasing the concentration to yield even better results. Atitration series (titrating amount of PE used) can be performed as well.In addition, optimization of the blocking procedure can improve results.

Example 9: Biotinylated Click Handled Lysate Conjugation

SNAPs prepared with a DBCO handle can be deposited on a chip surface andallowed to incubate sufficiently long to attach to the chip surface. Thechip can be then incubated for between 15 minutes and 30 minutes with ablocking buffer.

Lysate can be biotinylated and handled with an Azide click modifier. Thebiotinylated Lysate can be flown over the chip to allow clickconjugation of the Lysate to the DBCO SNAPs. The Lysate can then bedetected, for example via Streptavidin Lobe.

Example 10: DNA Origami

DNA origami SNAPs can be prepared for example with a DBCO handle, andcan be deposited on a chip surface and allowed to incubate sufficientlylong to attach to the chip surface. In some cases, the DNA origami SNAPscan be deposited on an array in a grid-like fashion. In some cases, theorigami SNAPs can be about 300 nm.

In some cases, DNA origami SNAPs can provide flexibility of SNAPorganization, shape, design, and sizing of the SNAPs compared with othertypes of SNAPs.

Example 11: Immobilization of Proteins from Lysate on Array

SNAPs were immobilized onto an array by flow. SNAP fluorescence wasdetected using a standard imaging protocol with a 100×100 micron fieldof view (FIGS. 18 A and 18 D). E. coli lysate comprising biotin handleswas applied to the array, and proteins were allowed to bind the SNAPs.As a control, SNAPs were exposed to and allowed to conjugate with lysatelacking the biotin handle. Fluorescence imaging was performed using astandard imaging protocol using fluorescent streptavidin, which can bindto biotin for detection, with a 100×100 micron field of view to detectthe lysate, As seen in FIGS. 18 B (biotin handle) and 18 E (control).Because the immobilized lysate does not contain the biotin tag, proteinsare not detected by fluorescent streptavidin. This control demonstratesthat the detection signal observed in (B) is specific to immobilizedproteins (i.e., that there is no non-specific binding of thestreptavidin detection reagent to the array surface).

The black and white images were multiplied to show co-localization ofSNAPs and biotin handled lysate in FIGS. 18 C (biotin handle) and 18 F(control). In the co-localization images, white indicatesco-localization, and black indicates no co-localization.

A fiducial can be seen in the bottom left corner of the image. HMIDSlanes without feature patterning can be seen as dark stripes on the topand right edges of the field of view. SNAPs on individual featureswithin the sub-array can be seen. Note: SNAPs are also easier tovisualize because of fluorescence cross-talk into this channel from thedetection channel (see B)

Example 12: Specific Detection of Short Peptide Epitopes (Trimers)

Fluorescently labeled SNAPs were immobilized onto an array by flow. Asmall peptide (HHH*) was allowed to conjugate with the SNAPs. As acontrol, SNAPs were immobilized to a chip and no peptide was conjugatedto the SNAPs. A fluorescent aptamer that is specific for the small HHHpeptide was applied. SNAP fluorescence was detected using a standardimaging protocol with a 35×35 micron field of view (FIGS. 19 A and 19D). This field of view can show the corner of one sub-array on a chip.The immobilized SNAPs can be seen as discrete spots on the array (eachof these spots is 300 nm in diameter).

Fluorescence imaging was performed in the same region in a differentfluorescence channel which can detect the aptamer, using a standardprotocol to detect the peptide (FIGS. 19 B (peptide) and 19 E (nopeptide control)).

The black and white images were multiplied to show co-localization ofSNAPs and the HHH peptide. FIGS. 19 C (peptide) and 19 F (no peptidecontrol) shows the co-localization of fluorescence between theSNAP-peptide channel and the aptamer detection channel. Co-localizationcan indicate successful binding and identification of the HHH peptide onSNAPs on the features of the array. Because there is no bound aptamer inFIG. 19 F, the merge image for the control image shows noco-localization.

In the no peptide control images, because the HHH peptide was unable tobind to the SNAPs on the array, there is no detection of HHH in theaptamer fluorescence channel. Thus, this result can confirm that the HHHpeptide was 1) directly attached to the SNAPs and 2) the signal observedin the aptamer channel can be observed only when the target peptide ispresent.

Two chrome fiducial marks can be seen in the lower right corner of theimage. The darker lanes in the image are HMIDS-coated areas that do notcontain patterned features.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of producing an array of differentproteins, comprising: (a) attaching a plurality of structured nucleicacid particles to a plurality of different proteins, such that each ofthe plurality of structured nucleic acid particles is attached to onlyone protein of the plurality of different proteins; and (b) attachingthe plurality of structured nucleic acid particles to an array ofattachment sites on a solid support, such that each of the array ofattachment sites is attached, via a structured nucleic acid particle, toonly one protein of the plurality of different proteins, therebyproducing the array of different proteins.
 2. The method of claim 1,wherein the plurality of structured nucleic acid particles comprisesnucleic acid origami.
 3. The method of claim 2, wherein the nucleic acidorigami comprises a long nucleic acid strand that is hybridized to aplurality of short nucleic acid strands.
 4. The method of claim 3,wherein the long nucleic acid strand is hybridized to at least 10 shortnucleic acid strands.
 5. The method of claim 3, wherein the long nucleicacid strand comprises at least 10 engineered folds.
 6. The method ofclaim 3, wherein the long nucleic acid strand comprises at least 100nucleotides.
 7. The method of claim 1, wherein the structured nucleicacid particle has a diameter between 50 nanometers and 100 micrometers.8. The method of claim 12, wherein the structured nucleic acid particlehas a diameter of at least 75 nanometers.
 9. The method of claim 1,wherein the proteins in the array of different proteins are separated byless than 1 micrometers.
 10. The method of claim 1, further comprisingdetecting proteins in the array of different proteins.
 11. The method ofclaim 1, wherein the plurality of structured nucleic acid particles isattached to the array of attachment sites through electrostaticinteractions.
 12. The method of claim 1, wherein the plurality ofstructured nucleic acid particles is covalently attached to the array ofattachment sites.
 13. The method of claim 1, wherein the plurality ofstructured nucleic acid particles occludes binding of more than oneprotein to individual attachment sites of the array of attachment sites.14. The method of claim 1, wherein (a) comprises covalently attachingthe plurality of structured nucleic acid particles to the plurality ofdifferent proteins, such that each of the plurality of structurednucleic acid particles is covalently attached to only one protein of theplurality of different proteins.
 15. The method of claim 1, wherein thearray of different proteins comprises 1000×1000 of the plurality ofstructured nucleic acid particles.
 16. The method of claim 1, whereinthe plurality of structured nucleic acid particles comprisesself-hybridized regions.
 17. The method of claim 1, wherein theplurality of structured nucleic acid particles comprises circularplasmids comprising a length of at least 5 kilobases.